Palisade pattern of mormyrid Purkinje cells: a correlated light and electron microscopic study.

THE JOURNAL OF COMPARATIVE NEUROLOGY 306~156-192 (1991)

Palisade Pattern of Mormyrid Purkinje Cells: A Correlated Light and Electron Microscopic-Study J. MEEK AND R. NIEUWENHUYS Department of Anatomy and Embryology, University of Nijmegen, 6500 HB Nijmegen, The Netherlands

ABSTRACT The present study is devoted to a detailed analysis of the structural and synaptic organization of mormyrid Purkinje cells in order to evaluate the possible functional significance of their dendritic palisade pattern. For this purpose, the properties of Golgi-impregnatedas well as unimpregnated Purkinje cells in lobe C, and C, of the cerebellum of Gnathonenus petersii were light and electron microscopically analyzed, quantified, reconstructed, and mutually compared. Special attention was paid to the degree of regularity of their dendritic trees, their relations with Bergmann glia, and the distribution and numerical properties of their synaptic connections with parallel fibers, stellate cells, “climbing” fibers, and Purkinje axonal boutons. The highest degree of palisade specialization was encountered in lobe C,, where Purkinje cells have on average 50 palisade dendrites with a very regular distribution in a sagittal plane. Their spine density decreases from superficial to deep (from 14 to 6 per pm dendritic length), a gradient correlated with a decreasing parallel fiber density but an increasing parallel fiber diameter. Each Purkinje cell makes on average 75,000 synaptic contacts with parallel fibers, some of which are rather coarse (0.45 pm), and provided with numerous short collaterals. Climbing fibers do not climb, since their synaptic contacts are restricted to the ganglionic layer (i.e., the layer of Purkinje and eurydendroid projection cells), where they make about 130 synaptic contacts per cell with 2 or 3 clusters of thorns on the proximal dendrites. These clusters contain also a type of “shunting” elements that make desmosome-likejunctions with both the climbing fiber boutons and the necks of the thorns. The axons of Purkinje cells in lobe C, make small terminal arborizations, with about 20 boutons, that may be substantially (up to 500 pm) displaced rostrally or caudally with respect to the soma. Purkinje axonal boutons were observed to make synaptic contacts with eurydendroid projection cells and with the proximal dendritic and somatic receptive surface of Purkinje cells, where about 15 randomly distributed boutons per neuron occur. The organization of Purkinje cells in lobe C, differs markedly from that in lobe C, and seems to be less regular and specialized, although the overall palisade pattern is even more regular than in lobe C, because of the absence of large eurydendroid neurons. However, individual neurons have a less regular dendritic tree, there is no apical-basal gradient in spine density or parallel fiber density and diameter, and there are no “shunting” elements in the climbing fiber glomeruli. Purkinje axonal boutons are not substantially displaced and have more but smaller boutons (on average about 701, which are not only contacting eurydendroid and Purkinje cells (about 40 boutons per cell), but also deeply located stellate neurons. As discussed in this study, none of the parameters analyzed is specifically and indissolubly correlated with the dendritic palisade pattern, and its functional significance consequently cannot be explained on the basis of a specific synaptic connectivity pattern. We suggest that palisade dendrites have a similar functional significanceas their spines and may be considered as super- or giant spines, subserving optimal tuning of mormyrid Purkinje cells for specific spatio-temporal patterns of parallel fiber activity. Comparison of different types of Purkinje cell

Accepted November 16,1990. Address reprint requests to J. Meek, Department of Anatomy and Embryology, University of Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.

o 1991 WILEY-LISS, INC.

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organizations as encountered in vertebrates shows two extremes: on the one hand, the mammalian configuration, probably specialized for optimal interactions with climbing fibers, and, on the other hand, the mormyrid palisade pattern, probably specialized for optimal interactions with parallel fibers. Key words: cerebellum, parallel fibers, climbing fibers, spines, stellate cells, Bergmann glia, teleosts,

Golgi-EM

The cerebellum belongs to the most thoroughly investi- cerebellum (Stendell, '14; Nieuwenhuys and Nicholson, gated and best understood regions of the vertebrate brain. '69a). The mormyrid cerebellum is relatively the largest one In particular, the mammalian cerebellar cortical organiza- encountered in the vertebrate kingdom: the overall braintion has been subject to detailed morphological and physio- body weight ratio is 150, comparable with that of humans logical research (e.g., Ramon y Cajal, '11; Eccles et al., '67; (Szabo, '83;Bell and Szabo, '86) and about 55%of this large Palay and Chan Palay, '74, '82; Ito, '84; King, '87). From mormyrid brain is encompassed by cerebellar tissue (Meek this research much insight has been gained in the proper- et al., '86b), yielding a cerebellum-bodyweight ratio of 1:90, ties of (1) granule cells and their excitatory mossy fiber a ratio much larger than in any other vertebrate. In input and parallel fiber output, (2) Purkinje cells with their particular the valvula cerebelli is huge and covers the dual input, i.e., from parallel fibers and from olivocerebellar complete dorsal aspect of the brain (Niewwenhuys and climbing fibers, and their inhibitory projection to central Nicholson, '69a). The mormyrid corpus cerebelli is differencerebellar nuclei, (3) several inhibitory feedback loops, as tiated into four lobes, numbered C,-C, (Nieuwenhuys and established by stellate cells in the molecular layer, by basket Nicholson, '69a; Meek et al., '86a), whereas the caudal part cells in the layer of Purkinje cells, and by Golgi cells in the consists of a granular eminence, with projections to the layer of granule cells, and (4)the parasagittal zonal organi- mechanosensory lateral line lobe (Maler, '74) and a lobus zation of the cerebellar cortex (e.g., Voogd, '67; Voogd and caudalis, connected with the electrosensory lateral line lobe Bigare, '80; Hawkes and LeClerc, '87). In nonmammalian of mormyrids (Maler, '74; Bell and Szabo, '86). Mormyrids amniotes, similar elements and organizational features are active electroreceptive teleosts, which means that they have been described,indicating a "basic cerebellar circuitry" have an electric organ and electroreceptors, and part of the (Llinas, '69) present in all amniotes. The amphibian cerebel- valvula and the lobus caudalis are involved in electroreceplum (Sotelo, '76; Llinas, '76) and the cerebellum of cartilag- tive or electromotor functions (Finger et al., '81; Bell, '86; inous fishes (Nicholsonet al., '69; Smeets et al., '83) seem to Bell and Szabo, '86). However, for the largest part of the valvula and the corpus cerebelli, there is presently no be organized similarly as well. The generally well developed cerebellum of teleostean evidence for an electroreceptive or electromotor function fishes shows several differences compared with the charac- (Meek et al., '86a,b), and consequently the functional teristics of the intrinsic and extrinsic cerebellar circuitry of significance of the mormyrid "gigantocerebellum" (Nieuthe remaining vertebrate groups indicated above. First, wehnuys and Nicholson, '69a) is still largely unknown. Apart from its size, the regularity of the molecular layer teleosts have a valvula cerebelli, a structure not present in other vertebrates. It is a cerebellar protrusion in the is a second remarkable feature of the mormyrid cerebellum. midbrain ventricle, located underneath the midbrain tec- This regularity is caused by the palisade pattern of the tum (e.g., Nieuwenhuys, '67; Finger, '83). Second, teleosts Purkinje cell dendrites, which all run parallel to each other, do not have central cerebellar nuclei, but instead euryden- perpendicular to the meningeal surface (Nieuwenhuys and droid projection cells located in the cerebellar cortex be- Nicholson, '69a,b; Nieuwenhuys et al., '74). Such a palisade tween the Purkinje cells (e.g., Nieuwenhuys and Nicholson, pattern is present throughout the valvula and the corpus '69b; Pouwels, '78a; Finger, '78b, '83;Meek et al., '86a,b; cerebelli, but not in the caudal lobe (Nieuwenhuys and Murakami and Morita, '87). Third, because of the presence Nicholson, '69a). A similar palisade pattern is present in of these eurydendroid projection cells instead of deep Xenomystis nigri, another osteoglossomorphteleost, which cerebellar nuclei, Purkinje cells do not project outside the has, however, a cerebellum of a "standard" teleostean size cerebellar cortex, but are interneurons with short axons (personal observation). Thus the regularity of the mormyrid terminating within the layer of Purkinje cells (Nieuwen- cerebellum is not directly correlated with its large size. The present work is devoted to a qualitative and quantitahuys and Nicholson, '69b; Nieuwenhuys et al., '74; Pouwels, '78a,b). Fourth, teleosts appear to have a peculiar tive analysis of the degree of regularity of the dendritic precerebellar nucleus lateralis valvulae not present in other palisade pattern of mormyrid Purkinje cells and the correlavertebrate groups. This nucleus gives rise to a massive tion of this regularity with other morphologic and synaptic mossy fiber projection, not only to the valvula, but also to organizational features of these cells. The aim is to explore the teleostean cerebellar corpus (Finger, '78a, '83;Meek et the possible functional significanceof the palisade organizaal., '86a,b; Wulliman and Northcutt, '88, '89). In many tion of the mormyrid molecular layer. In particular lobe C, other respects, the teleostean cerebellum is comparable to and C, were selected for analysis and mutual comparison as that of other vertebrates, containing granule cells, Purkinje these lobes represent two extremes in the variability encouncells, Golgi and stellate neurons, mossy, parallel, and tered, as will become clear from the results. In addition, the climbing fibers (Larsell, '67; Nieuwenhuys, '67; Paul, '82; connections of these lobes are well known (Meek et al., Finger, '83;Nieuwenhuys and Pouwels, '83). It should be '86a,b), and several morphological aspects of these lobes have been described qualitatively in previous studies (Nieunoted that basked cells are absent in teleostean cerebella. Within the group of teleosts, mormyrids are of special wenhuys and Nicholson, '69a,b; Kaiserman-Abramof and interest because of their extremely large and highly regular Palay, '69; Nieuwenhuys et al., '74; Nieuwenhuys, '76). A

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preliminary report has been presented at the sixteenth Neuroscience Meeting (Meek and Nieuwenhuys, '86).

MATERIALS AND METHODS Preparation of tissue The present study is based on histological material obtained from 28 specimens of Gnathonemus petersii, ranging from 10 to 15 cm in length. For Golgi impregnation and subsequent LM or EM investigation, the following procedure was used. After anaesthesia with MS222 (0.1 g/l, 5 min.), the skull was removed to expose the brain. Within the next minute, the animal was sacrified by transection of the medulla oblongata, and various dissected parts of the cerebellum, including lobe C, and lobe C,, were immersed in a fixative for 3 hours at room temperature. The fixative consisted of different concentrations of aldehydes in 0.1 M phosphate buffer, varying from 2% paraformaldehyde and 2% glutaraldehyde to 5%of both aldehydes. Best results were obtained with the higher concentration (4% or 5% of both aldehydes), but other fixations yielded useful additional results, with different cell types impregnated or different numbers of impregnated elements. Following prefixation with aldehydes, the Golgi-rapid procedure was applied, including 3 days immersion of tissue at room temperature in a mixture of 0.2% osmiumtetroxide and 2% potassium dichromate in distilled water followed by 2 days immersion at room temperature in 0.75% silver nitrate. Next, 50-pm sagittal serial sections were cut on a vibratome, the trough being filled with 50% ethanol saturated with silver chromate (0°C; see Blackstad, '75; Meek, '81a). The sections obtained were quickly studied in the light microscope to select material for the deimpregnation procedure and subsequent preparation for electron microscopy. Sections not selected for further processing were made suitable for LM analysis by dehydration with ethanol and xylene, mounting on slides in depex mounting medium, and coverslipping. They were used to study impregnated cerebellar elements light microscopically with the aid of a Leitz light microscope with a drawing tube. For electron microscopy, selected vibratome sections were treated according to the deimpregnation procedure of Fairen et al. ('771, which included gold toning for 15 minutes in 0.05-0.1% hydrogen tetrachloroaurate (HC1, AuH,O), at 5-10°C, washing in distilled water, immersion for 5 minutes in 0.05% oxalic acid at 5-10°C, washing in distilled water and deimpregnation for 3 0 4 5 minutes in 1% sodium thiosulfate at 20°C. After several washes, deimpregnated sections were postfixed for 30 minutes in 1% osmium tetroxide in phosphate buffer, dehydrated in a graded series of ethanol and via propyleneoxyde embedded in Epon 812, between a slide and a coverslip protected with repelcote to facilitate remounting. After polymerization of the Epon 812, deimpregnated sections were studied in the light microscope, and cells of interest were drawn and selected for further processing, including removal of the repel-coated slide and coverslip, dissection of a small part of Epon containing only the cell of interest, remounting of this cell on a prepolymerized Epon block with the aid of liquid Epon, which then was allowed to polymerize, and final trimming and ultrathin sectioning. Serial sections of white interference colour (60-90 nm) were cut and mounted on 50-mesh copper grids covered with a formvar-film. On each grid a fixed number of sections per series was mounted,

ranging from 5 to 10 per series. After section of 50-80 sections, the pyramid was studied under the light microscope with transmitted light from below, to investigate which parts of the cell of interest had disappeared and thus should be located in the previous 50-80 sections, which substantially facilitates the electron microscopical analysis. The ultrathin sections were contrasted with uranyl acetate and lead citrate and studied in a Philips EM 301. For comparison and a number of morphometric measurements, several additional brains were used. Some of these were treated in a similar manner to the procedure described above, however, omitting the Golgi-impregnation and deimpregnation procedure. Others were obtained from fishes that were first very shortly perfused with saline (0.9% NaCl), followed by 1-minute perfusion with 2% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer and then 10 minutes perfusion in 4% glutaraldehyde and 5% paraformaldehyde in the same buffer. Both ultrathin and semithin sections were obtained from this material, not only in the sagittal direction, but also in the horizontal and transverse plane.

Electron microscopical analysis and reconstruction To study the synaptic properties of cells of interest, first, electron micrographs were made at low magnification (about 2,00Ox), of about one of each 5 to 20 sections of a series. Second, photomontages were made including all parts of the neuron under investigation. The contours of relevant elements were drawn on a transparent sheet, and the location of synapses and other specializations were indicated on these drawings. Next, the sections used for the photomontage were reinvestigated at higher magnification in the electron microscope to study details of the synaptic configurations. In this stage, additional sections, located in the series of ultrathin sections under study between the previously selected sections, were studied and photographed as well when this was necessary to make detailed reconstructions of, e.g., the axon hillock or clusters of climbing fibers, or to extend the number of drawings available for reconstruction or the number of photographs necessary for quantifications. Reconstructions of cells, made by hand from the drawings made at 2,000 times magnification, were used to indicate the distribution of the synaptic contacts observed in the electron microscope on the receptive surface of the reconstructed cells (see Figs. 17, 18). Apart from the deimpregnated neurons selected, several unimpregnated neurons present in the section studied were analyzed and reconstructed as well.

Morphometric measurements and calculations By means of morphometric methods, an estimation of the order of magnitude of several parameters was obtained, including the size and number of granule cells, Purkinje cells, and parallel fibers per cerebellar lobe studied; and the number and dimensions of dendrites, dendritic spines, axonal boutons, and synaptic contacts of various origin per Purkinje cell in the lobes studied. A first approximation of several of these parameters was simply derived from countings and measurements applied on drawings of wellimpregnated Purkinje cells, such as an estimation of the number of dendrites and spines per Purkinje cell, the axonal and dendritic dimensions of these neurons, and the number and dimensions of axonal boutons. To have drawings of complete neurons, always light microscopical serial

PALISADE PATTERN OF MORMYRID PURKINJE CELLS sections (of 50 pm) were used to ascertain that a cell was indeed restricted to a single section or to study additional parts of the cells under study in neighbouring sections. For most measurements and calculations, semithin (1 pm) or ultrathin (60-90 nm) sections were used. In such sections accurate countings can be obtained for the presence of sectioned profiles of a variety of structures, from which estimations of their volume densities or actual numbers can be obtained by means of stereological formulae. In the present study we used basically the AbercrombieFloderus formulae, with a separate approach for spheroid particles (nuclei and spines) and disklike structures (synapses). For nuclei and spines the Abercrombie-Floderus formula reads: N* (Floderus, '44;Abercrombie, '461, (I) N, = D+t-2h in which NA is the number of particle profiles counted per surface area of section; D is the caliper factor or the height, perpendicular on the section direction, of the counted particles; t is section thickness (1 km for semithin sections and 80 nm for ultrathin sections), and h is the height of lost caps, i.e., caps of nuclei or spines not recognized in the sections used for counting. was calculated according to the formula given by Smolen et al. ('831, which reads:

[

D = d 1-

-

I

(1 - 4/-rr)d

t+d

(Smolen et al., '83),

(2)

in which a is the mean diameter of particle profiles in the sections used; h was determined from the radius of the smallest profiles visible, by the formula given by Weibel ('79): h = R - JR2 - ro2 (Weibel, '79),

(3)

in which R represents the mean particle radius, and ro the radius of the smallest profiles visible in the sections analyzed. A detailed evaluation of these formulae was given by us previously (Albers et al., '88). For calculation of the density and number of synaptic contacts, which were considered as round disks, we used the formula, according to Colonnier and Beaulieu ('85) and Albers et al. ('go), which reads:

N,

=

Na/L.

(4)

This is basically the same formula as explained above for spheroid particles, since it has been shown that the caliper factor for synaptic contacts equals their mean trace or chord length (L ) in ultrathin sections (Kaiserman-Abramof and Peters, '72; Mayhew, '79; Colonnier and Beaulieu, '851, whereas h equals l/zt in such material, and thus t - 2h becomes zero (Meek, '81b; Albers et al., '90). In a test system, formula (4) has been shown to yield the most consistent and valuable results of a variety of formulae tested (Colonnier and Beaulieu, '85). To apply the formulae discussed above, we counted particles of interest (nuclei, spines, and synaptic contacts) in samples selected by superposition of a test lattice with forbidden lines (Gunderson, '77) over the micrographs or drawings used. Diameters of nuclei and spines were determined by projection of a measuring grid over the picture

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observed in the light microscope or over the micrographs used, respectively, whereas trace lengths of synaptic contacts were measured with a Kontron-Videoplanequipment. The magnification factors in LM measurements were determined with an object micrometer, and in EM measurements with the aid of a replica of 2,160 lines per mm. A similar approach was used to determine the diameters of parallel fibers in sagittal sections, and to determine the dimensions of the palisade pattern in the molecular layer in transverse, sagittal as well as tangential semithin and ultrathin sections, the latter running parallel to the surface of the molecular layer and thus perpendicular to the palisade pattern. In detail, the following measurements and calculations were applied, based on the formulae and considerations presented above. Sometimes advantage was taken of the orthogonal, regular organization of the molecular layer. Unless otherwise indicated, random samples from sections in different directions of different animals were used (see Tables 1-3). 1. Dimensions of lobe C, and C, (a) The width (lobe C,) or circumference and surface area (lobe C,) of the molecular layer and the granule-cell layer was determined using transverse sections of 50- or 1-km thickness of a variety of animals. (b) The total, unfolded rostrocaudal length of the surface of the molecular layer and the layer of Purkinje cells (i.e., the surface of the granule cell layer) was determined in sagittal sections of lobe C, and C, close to the midline. (c) The volume of the granule cell layer of lobe C, was determined by considering the granule cell mass as a cylinder with a spherical cap, from which the volume can be calculated by mathematical formulae from the measurements obtained under l a and b. The percentage of the surface area occupied by myelinated fiber bundles in transverse sections was subtracted from this value. (d) The volume of the granule cell layer of lobe C, was determined by measurements of the surface area of this granule cell layer in a large number of sagittal sections in a variety of animals (Table 1) and multiplication of this area with the width as determined under la. (e) The surface of the molecular layer and granule cell layer (the latter also representing the surface of the Purkinje cell layer) was determined for lobe C, by multiplication of the length and width determined under (a) and (b). For lobe C, a similar approach was used as indicated under (c), i.e., by formulae for a cylinder with a spherical cap. ( f ) The height or thickness of the molecular layer, corresponding to the average length of palisade dendrites, was measured in the same sections used under (a, and (b). 2. Number of granule cells per lobe (a) N, (i.e., the numerical density of granule cells per surface area of sections) was determined in a number of semithin sections of different animals in different directions (Table 21, from which random samples were analyzed. (b) d (i.e., the mean diameter of nuclear profiles) and r, (the radius of the smallest nuclear profiles recognized) were determined for the same samples.


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3.

4.

5.

6.

(c) N, (i.e., the numerical volume density or number per unit volume) was calculated by means of formulae (l),(2),and (3). (d) The number per lobe (C, and C,) was estimated by multiplication of the result of (c) with l(d) or l(c), respectively. Number of Purkinje cells per lobe Basically, a modified formula (1)was used, since the layer of Purkinje cells is a monolayer, and we are not so much interested in N, but in N, (the number of Purkinje cells per surface area of the Purkinje cell layer) and consequently did not determine N,, but instead N, (i.e., the numerical density of Purkinje cells per unit length of Purkinje cell layer present in the sections). (a) N, was determined in semithin sections in the transverse and sagittal direction. (b) a and r, were determined in the same sections for the same sample. (c) N, was estimated by means of formulae (21, (31, and a modified formula (11, in which N, was replaced by N, and N, by N,. (d) The number of Purkinje cells per lobe was calculated by multiplication of the result of (c) with l(e) both for lobes C, and C,. Number of Purkinje dendrites per lobe (a) The dimensions of the palisade pattern, i.e., the mean center-center distance between palisade dendrites in the transverse and sagittal direction, was determined in tangential, sagittal, and transverse sections. (b) On the basis of (a), the surface density of Purkinje dendrites per surface area of a tangential section was determined. (c) The number of dendrites per lobe (C, or C,) was estimated by multiplication of the result of (b) with the mean of the surface area of the granule and molecular layer of each lobe as estimated under l(e). Number of palisade dendrites per Purkinje cell (a) A first approximation was obtained by counting the number of dendrites of the best Golgi-impregnated Purkinje cells in the material available. These cells were also used to measure the rostrocaudal extension of the dendritic tree of Purkinje cells. (b) A second estimation was derived from estimations of the number of palisade dendrites and Purkinje cells per lobe, which means by division of the result of 4(c) by the result of 3(d). Number of spines per palisade dendrite and per Purkinje cell (a) Preliminary estimations were obtained by counting spines in a large number of palisade dendrites of Golgi-impregnatedneurons, usinga lOOx oil immersion objective lens. (b) A second estimation was based on measurements and calculations using tangential ultrathin sections at a superficial, intermediate, and deep position in the molecular layer of lobe C, or lobe C,. (i) The number of profiles of spines and (perpendicularly running) Purkinje dendrites was counted, and the ratio between counted spines and dendrites (N,/Den) was calculated. (ii) The caliper factor of spines was calculated on the basis of measurements of the average

diameter H, and the smallest recognizableprofile radius r,. (iii) Formulae (l),(Z),and (3) were used to estimate the volume density of spines per dendrite (NJDen), which corresponds to the number of spines per unit length of palisade dendrite, because of the use of tangential sections and the orthogonal course of Purkinje dendrites. (iv) The average number of spines per dendrite was estimated by multiplication of the average result of 6(biii) with the average length of palisade dendrites as determined under l ( f 1. (v) The average number of spines per Purkinje cell was estimated by multiplication of the number of spines per dendrite Gtbiv) with the number of dendrites per cell 6(a)or 5(b). 7. Number of parallel fiber contacts per palisade dendrite and per Purkinje cell (a) The number of synaptic contact profiles and (perpendicularly) sectioned palisade dendrites was determined in the same tangential sections used under 6(bi), and their ratio was calculated (i.e., N,,/Den). (b) The caliper factor (= the mean trace length was determined for the same contacts. (c) The volume density of synaptic contacts per dendrite was determined using formula (41, in which N, was replaced by N,/Den and thus N,/Den is calculated instead of N,, which means the number of synaptic contacts per unit length of dendrite; see also G(biii). (d) The total number of parallel fiber contacts per palisade dendrite was estimated by multiplication of the result of (c) with that of l(f 1. (e) The number of parallel fiber contacts per Purkinje cell was estimated by multiplication of the result of 7(d)with that of 5(a)or 5(b). ( f ) Multiplication of the mean trace length see 7(b)) with 417 yielded an estimation of the average diameter of parallel fiber synaptic contacts with spines (Hilliard, '67). 8. Number and diameter of parallel fiber profiles in sagittal sections (a) In sagittal ultrathin sections of lobe C, and C,, the number of transversely sectioned parallel fiber profiles per 10 km height per parallel fiber septum was counted in a number of samples at a deep, intermediate, and superficial position in the molecular layer. (b) The number of parallel fiber profiles per sagittal section of a parallel fiber septum was determined by multiplication of the average value obtained under (a) with the average thickness of the molecular layer l(f). (c) An estimation of the number of parallel fiber profiles in a sagittal section of the complete lobe C, and C, was obtained by multiplication of number of fibers per septum (b) with the total number of septa per lobe, which equals the unfolded length of the molecular layer of lobe C, and C, (l(b)),divided by the sagittal palisade pattern distance (4(a)). (d) The same sample indicated under &a) was used to determine average diameters and diameter spectra of parallel fibers at a deep, intermediate, and superficial position in the molecular layer.

z)

(z;

PALISADE PATTERN OF MORMYRID PURKINJE CELLS 9. Length of parallel fibers (a) In transverse sectioned Golgi-preparations, the length of the best-impregnated parallel fibers was measured. In lobe C,, these appeared to traverse the entire width of this lobe, whereas in lobe C, they were as long as about half of the circumference of this lobe. (b) A second estimation of the length of parallel fibers was obtained from the ratio between the number of parallel fiber profiles in sagittal sections of lobe C, or C, (8(c))and the number of granule cells per lobe (2(d)). This ratio should be 1 for lobe C,, when parallel fibers all have the same width as the molecular layer, and becomes smaller when parallel fibers are on average shorter. In lobe C, a ratio of 1would mean an average length equal to half of the circumference. Consequently, multiplication of the ratio 8(c)/Z(d)with the width or half circumference of lobe C, and C,, respectively, yields an estimation of the average length of parallel fibers in a lobe. It should be noticed that this approximation is based on the assumption that each granule cell gives rise to one parallel fiber, not more and also not less. 10. Density and number of synaptic contacts per parallel fiber (a) A first impression was obtained from transversely sectioned Golgi impregnations (cf. 9(a)). (b) A second estimation was obtained as follows: (i) The percentage of parallel fibers in a septum that contacts a passed Purkinje dendrite is given by the ratio between the number of synaptic contacts per palisade dendrite (7(d)) and the number of parallel fibers per septum (8(b)). (ii) The ratio calculated under (bi) equals the ratio between the number of Purkinje cell dendrites that are and are not contacted by each traversely running parallel fiber. (iii) Combination of the ratio calculated under (bii) and the transverse dimensions of the palisade pattern (4(a)) yielded an estimation of the average distance between neighbouring synaptic contacts of a parallel fiber, or the density of these contacts per parallel fiber, which means the average number of synaptic contacts per unit length of parallel fiber. (iv) The average number of synaptic contacts per parallel fiber is estimated by multiplication of the density calculated under (biii) and the average length of parallel fibers (9(a) or 9(b)). 11. Number of climbing fiber contacts per Purkinje cell (a) The number of climbing fiber synaptic contact profiles (NJ, recognized and identified as indicated in Results, was counted in one out of each 5 to 8 serial sections of the series used to reconstruct Purkinje cells. (b) The caliper factor (i.e., the mean trace length E ) was determined for these contacts with a KontronVideoplan equipment. (c) The number of contacts per cell, N, was subsequently calculated using formula (41, in which N, was substituted by N and NAby N,, the result of which has to be multiplied by 5 to 8 times .08 pm (section thickness), depending on the selection of 1 out of 5 or 8 sections for the countings under (a),to

161

yield the ultimate number of climbing fiber synaptic contacts per Purkinje cell. (d) An estimation of the average diameter of climbing fiber synaptic contacts was obtained by multiplication of E (b) with IT (see 7(f)). (e) The number of climbing fiber clusters or glomeruli per cell was counted in the reconstructions of cells made from serial sections (see figs. 17, 18). 12. Number of Purkinje axonal boutons per axon This parameter was counted in sagittal sections of Golgi preparations, using the best impregnated examples in lobe C, and C,. 13. Number of Purkinje axonal boutons contacting a Purkinje cell This number was counted on the reconstructed cells that were also used to determine the distribution of climbing fiber clusters as described above (ll(e); see Figs. 17, 18).

RESULTS Gross morphology and morphometry of the lobes C, and C, Lobes C, and C, differ substantially in their gross morphology and represent two extremes in the variability encountered in the mormyrid corpus cerebelli. Lobe C, is essentially a narrow sheet of cerebellar cortex, flat in the transverse direction and rostrocaudally curved around the commissure of lobe C , (Fig. 1).The width of the ganglionic layer, i.e., the layer of Purkinje and eurydendroid cell bodies, is on average 700 pm and the unfolded rostrocaudal length is on average about 4,300 pm (Table 1; Fig. 1). The width of the molecular layer is similar, but that of the granule layer is somewhat larger (800 pm) since granule cells are not only located “underneath” the ganglionic layer, but also lateral to the ganglionic and molecular layers (Figs. 1, 2). The cortex of lobe C, can be subdivided in a rostral, dorsal, and caudal part on the basis of the relative position to the central commissure (Fig. 1).The dorsal part has in small specimens a transverse fissure, about in its center (Fig. 1). In contrast to lobe C,, lobe C, establishes a true lobe, with a caudal peduncle and a free rostral pole. In transverse sections (Figs. 1,2),lobe C, is rostrally circular and caudally more flattened. The unfolded width of the ganglionic layer, i.e., its circumference, in on average about 2,500 p,m and the surface area of the granule cell layer is on average about 0.4 mm’. The length of lobe C, as a whole, measured from the rostral pole of its peduncle to the rostral cap of molecular layer, is on average 1,800 pm (see Table 1). A marked difference between lobe C, and C,, already obvious at low magnification, concerns the occurrence of myelinated fibers in the ganglionic as well as the molecular layer. Lobe C, contains many myelinated parallel fibers in the lower molecular layer, and also many myelinated axons at the transition zone between the ganglionic and granular layer (e.g., Fig. 4A). In lobe C,, myelinated fibers in the ganglionic layer and the molecular layer are scarce. To obtain an estimation of the order of magnitude of the number of Purkinje cells and granule cells in both lobe C, and C,, some morphometrical methods were applied (Table 2). The surface density (i.e., the number per unit of surface ganglionic layer) of Purkinje cells in both lobe C, and C, is


162

*I

"I C

I

E

,I

GI

Fig. 1. Drawings of sagittal (A) and transverse (B-G) sections through the corpus cerebelli of Gnathonernuspetersii to show the shape of lobe C, (A, E-G) and lobe C, (A-D). The level of sections B-G is indicated in A. Cerebellar lobes are indicated as C,, C,, C,, and C,. The stippled regions indicate the granular layer; the molecular layer of lobe

E

I

F

G

C, and C, is subdivided in the following regions: c: caudal region (of CJ; d: dorsal region (of C, or CJ; r: rostral region (of C, or CJ; v: ventral region (of CJ. Further abbreviations indicate: cC,: commissure of C , ; co: commissure; It: lobus transitorius (between corpus and valvula cerebelli); pC,: peduncle of lobe C,. Magnification: x22.

Fig. 2. Photographs of transverse sections through the brain of Gnathonernus petersii to show the position of lobe C, (on the left side) and lobe C, (on the right side). For furhter details see Meek et al. ('86a). Magnification: x 12.

on average 2,300/mm2.Since the surface of the ganglionic layer was estimated to be 3.0 mm2in lobe C, and 3.8 mm2in lobe C,, the estimated average number of Purkinje cells in lobe C, is about 7,000 and in lobe C, about 9,000 (Table 3). The number of 7,000 Purkinje cells in lobe C, is of the same order of magnitude as the number of 6,866 calculated by Nieuwenhuys et al. ('74) using completely different methods. The volume density of granule cells he., their number per volume unit) is in lobe C, on average about 5 per 1,000

pm3, without a marked difference between rostral, dorsal, or caudal regions in our sample (Table 2). The numerical density of granule cells in lobe C, is substantially higher, about 12 per 1,000 pm3 in our aldehyd-osmium fixed material. By multiplication of the density with the volume of the granule cell layer of lobe C, and lobe C, (0.43mm3and 0.58 mm3, respectively), an estimation of about 2 million granule cells in lobe C, and about 7 million in lobe C, may be calculated. It should be noticed that these estimations are only meant to give an impression of the order of magnitude


163

TABLE 1. Dimensions of Lobe C, and C, in Sagittal Sections Close to the Midline

c3

Cl

~

Fish length (cm)

Fish nr. G5 G6 GI G8 G9 G10 G11 G12 G13 G14 Average

12 10 9 15 11 12 9 14 14 13 11.9 _i 2.13 (10)

Length of ganglionic layer

Surface of granule cell layer

ipm)

(pm2)

3,707 3,541 4,450 4,024 4,775 3,550 4,725 5,275 4,733 4,309 & 625 (9)

359,374 456,597 626,736 460,067 574,652 407,984 635,416 868,055 467,013 539,544 ? 155,793 ( 9 )

Length of granule cell layer (iLm) 1,525 1,460 1,382 1,775 1,500 1,583 1,875 1,300 1.550 & 192 ( 8 )

TABLE 2. Nuclear Diameters and Numerical Densities of Granule- and Purkinje Cells in Lobe C, and C, Granule cells

GI G2 G3 G4 G15 G16 Average value

Section direction sag Sag Sag Sag Transv Transv

D’

(pm)

NY2 (per 1,000 pm3)

4.4 2 0.3 (41 4.6 (1)

4.32 2 0.43 (4) 4.37 (1)

4.0 2 0.2 (41 4.1 2 0.2 (11) 4.3 & 0.3 (4)

6.33 f 1.09 (41 5.72 t 0.93 (111 5.19 + 1.00 (4)

c,

c3

Cl Animal

Purkinje cells

D

D

Ns3 (per mm’)

D (pm)

1.4 (31

2,800 5 1,880 (3)

8 6 2 0 . 9 (21 8.6 2 1.0 (3)

1,600 -c_ 10 (21 3,370 2 100 (3)

6.7 (11 8.1 i 0.5 (51 8.2 -r 1.6 (3)

1,560 (1) 2,530 2 1,020 (51 2,300 i 650 (31

9.6 2 0.5 (2) 6.6 2 0.4 ( 6 ,

1,500 -c 40 (21 2,780 & 1,050 ( 6 )

8.5 i 1.2 (4)

2,310

Nv

(pm)

(per 1,000 pm3)

3.3 i 0.0 (2) 4.2 i 0.1 (4) 3.9 ? 0.4 (31 3.7 I 0.4 (4) 3.6 I 0.1 (11)

15.54 t 1.12 (21 8.69 i 0.56 (4) 11.53 i 1.98 (31 11.29 i 3.36 (4) 14.20 i 2.88 (111

3.7 ? 0.3 (5)

12.25 i 2.68 (51

c3

(pm) 9.8

t

NS

(per mm’)

i 910

(4)

‘Diameter. ‘Number per volume unit. 3Numberper surface area.

and that substantial variations may occur in different animals, partly correlated with their size (Tables 1 , Z ) .

Gross features of mormyrid Purkinje cells Mormyrid cerebellar Purkinje cells resemble those of other vertebrates in the sagittal orientation of their dendritic tree, perpendicular to the transversely running parallel fibers. However, they differ from most other Purkinje cells in their dendritic branching pattern, since the smooth proximal dendrites do not protrude into the molecular layer, but instead give rise to dendritic tufts at the boundary between the ganglionic and the molecular layers, from which long, mostly unbranched, very spiny dendrites originate, running parallel to each other and oriented perpendicular to the ganglionic layer and the meningeal cerebellar surface (Fig. 3). A second marked difference compared to most other vertebrates, except for other teleosts, is the fact that axons of mormyrid Purkinje cells do not project outside the cerebellar cortex to central cerebellar nuclei, which do not exist in mormyrids (Meek et al., ’86a,b). Instead, they remain within the ganglionic layer to terminate mainly on other Purkinje cells and eurydendroid neurons. The latter represents teleostean cerebellar output cells with cell bodies located in the ganglionic layer, dispersed among those of the Purkinje cells (Nieuwenhuys et al., ’74; Finger, ’78b; Pouwels, ’78a,b; Meek et al., ’86a,b; Murakami and Morita, ’87). In the following section, the morphology and synaptology of the Purkinje cells in lobe C, and C, of the mormyrid cerebellum is described in detail, going roughly from apical to basal. Thus first the apical spiny palisade dendrites and their relations with Bergmann glia, parallel fibers, and stellate cells are described. Second, the proximal smooth dendritic trunks and cell bodies and their contacts with

olivocerebellar ‘‘climbing”fibers are dealth with. Last, the morphology and distribution of Purkinje axonal arborizations and terminations are quantified.

The palisade dendrites in the molecular layer The most regular orthogonal organization of the apical dendritic trees of individual mormyrid Purkinje cells in encountered in lobe C,. In this lobe, the best Golgiimpregnated examples of Purkinje cells reveal 50-60 apical spiny dendrites. Calculations dividing the estimated total number of dendrites in lobe C, (see below) by the estimated number of about 7,000 Purkinje somata (see above) suggest a similar average number of about 50 dendrites per Purkinje cell. The dendrites of single neurons are generally quite strictly located in a single sagittal plane (Fig. 4A,D) and fill this plane regularly, without many empty columns for dendrites of other cells. Only 8% of the palisade dendrites gives rise to a secondary palisade dendrite originating in the upper 2/3 of the molecular layer, whereas the remaining 92% is unbranched from the lower molecular layer to the meningeal surface. The observation that in Golgi preparations many dendrites do not reach the meningeal surface (e.g., Fig. 3) is an impregnation artefact, as demonstrated by Golgi-EM and normal EM analysis. Consequently, the extrapolated reconstructions drawn in Figure 5 show the organization of the apical dendritic tree of Purkinje cells in lobe C, most realistically. The apical dendrites of mormyrid Purkinje cells are densely packed with spines (Fig. 4D). In lobe C,, we could observe in Golgi preparations on average about 75 spines per 25 pm dendritic length, without significant differences comparing rostral, caudal, or central dendrites, but with a significant decrease from apical to basal parts of the dendrites (from 92 to 53). Starting from an average length


164

Fig. 3. Drawing of a typical Purkinje cell in lobe C,, showing the palisade pattern of the spiny distal dendrites, the smooth proximal dendrites and cell body, and the displaced terminal arborization of the axon in the ganglionic layer. Magnification: 325.

of a palisade dendrite of 200 Km (i.e., the average thickness of the molecular layer), this would mean a number of about 600 spines per dendrite and about 30.000 per neuron. Electron microscopicalmeasurements, however, reveal that this is a serious underestimation, obviously caused by masking of spines by the Golgi precipitate in the palisade dendrite and in nearby spines. Electron microscopical measurements suggest an average number of about 2.000 spines per dendrite and thus about 100.000 spines per neuron in lobe C, (Table 3), with a significant decrease in spine density from superficial to deep in the molecular layer (see below). In Golgi preparations of lobe C,, several quantitative differences in the organization of the apical dendrites of Purkinje cells were noticed compared to lobe C, (Table 3 ) . The estimated mean number of dendrites per neurons in lower 6 e . , 30-50 in the best Golgi-impregnated examples, and about 30 in the dorsal part of lobe C, as calculated by division of the number of dendrites by the number of Purkinje cells); the dendrites of a single neuron are frequently located in more than one sagittal plane; and 25%of the palisade dendrites gives rise to a secondary or tertiary branches within the molecular layer, whereas only 75% remains unbranched from the lower molecular layer to the meningeal surface. Thus Purkinje cells in lobe C, give the impression to be less strictly organized than in lobe C,, an impression supported by the organization of the proximal dendritic tree, which shows many “crossing overs,” in contrast to those in lobe C, (see below). The average density of spines counted in Golgi preparations in lobe C, is about

80 spines per 25 pm dendritic length. Like in lobe C,, calculations based on electron microscopicalsections yielded much higher numbers, i.e., a density of 14 per Km dendrite, which means about 3,000 spines per dendrite and about 80.000 spines per Purkinje cell, a number comparable to that calculated in lobe C,. In contrast to lobe C,, the dendrites in lobe C, had no significant or consistent differences in apical or basal spine density. In the electron microscope, both lobe C, and C, show a very regular orthogonal palisade pattern (Fig. 6). In the sagittal direction, the Purkinje dendrites are separated by septa of parallel fibers, and in the transverse direction by processes of Bergmann glial cells (Fig. 7). Bergmann glial cells have their cell bodies preferentially in the boundary region between the ganglioniclayer and the molecular layer (Fig. 7). In lobe C, they even constitute almost a separate cellular layer in this region, with the effect that a pronounced glial barrier is present between the ganglionic and the molecular layer (Fig. 8D). Each Bergmann cell gives rise to a number of long, apical processes, which, like t,he Purkinje dendrites, are mostly unbranched throughout their course from deep to superficial, and thus equally establish a palisade pattern. However, their apical processes are not restricted to the sagittal plane, but are roughly located in a cylindrical region above their parent cell body (cf. Fig. 7). The dimension of the palisade patterns in lobe C, and C, are presented in Table 3. In lobe C,, in the sagittal direction about 32 Purkinje dendrites per 100 p,m are encountered, and in the transverse direction about 40, which means an


- - - -

~

Figure 4. For legend see overleaf.

165


166

Fig. 5. Drawings of the dendritic tree of Purkinje cells in lobe C,, to show their regularity and variability. Stippled dendrites are extrapolated from incomplete impregnation. Notice the absence of dendrites that cross each other in the sagittal plane, and the low number of distally branching palisade dendrites. Magnification: x 190.

average center to center distance of 3.1 and 2.5 pm respectively. In lobe C, these distances are on average larger, i.e., 4.4 pm in the sagittal and 2.9 pm in the transverse direction (Table 3). At several places, the regular palisade pattern of the Purkinje dendrites and Bergmann glial processes is interrupted by ascending bundles of granule cell-axons (Fig. 6C) and by dendrites of large eurydendroid cells. The small dendrites of stellate cells or of the small eurydendroid cells in lobe C, do not substantially disrupt the palisade pattern, but intermingle with it in a more subtle way. At the meningeal surface, the end-feet of

Fig. 4. Photomicrographs of Golgi-impregnated Purkinje cells in sagittal sections. A. Two impregnated neurons in lobe C,, with a regular palisade pattern. Arrowheads point to the long axon of the left neuron. B. Detail of the left cell in A, showing the smooth proximal dendritic tree that gives rise to spiny dendritic tufts. Arrow points to thorns that are presumed to make contact with climbing fiber terminals. C . Initial part and terminal arborization of the axon of a Purkinje cell in lobe C,. D. The distal palisade dendrites of a Purkinje cell in lobe C, with their high density of spines. Magnifications: A: ~ 2 7 5B,C,D: ; x 1050.

the Bergmann glial processes cover the distal tips of the Purkinje cells, whereas the parallel fiber septa at the meningeal surface are covered by a distinct population of glia cells (Fig. 6E,F). Quantitative analysis of electron microscopical pictures suggests that Purkinje dendrites in lobe C, bear on average as much as about 9 spines per pm length, with a significant decrease from superficial levels (about 14 per pm) via intermediate (about 10 per pm) to deep levels (about 6 per pm) in the molecular layer (Table 3). The average spine diameter in lobe C, was about 0.4 pm. In lobe C, the average spine density is even higher, about 14 per pm dendritic length, however, without a decrease from superficial.Spines in lobe C, seem to be slightly smaller than in lobe C, (about 0.35 pm). It should be noted that a large variability exists in spine densities, varying from 10-19 in lobe C, and even from 4 (deep) to 17 (superficial)in lobe C,.

Synaptic contacts with parallel fibers As described above, the palisade pattern of the mormyrid cerebellar molecular layer consists of rows of alternating

PALISADE PATTERN OF MORMYRID PURKINJE CELLS TABLE 3. Quantitative Asuects of Mormvrid Purkinie Cells

c,

C3

CELL BODY Number per lobe (semithin)” 6,925 t 2,200 (9)’’ 8,800 t 1,300 ( 8 ) Diameter (Golgi) 26 2 5 pm (151 22 2 4 pm (12) Number of primary dendrites (Golgil - mean 2.8 i 1.0 (231 1.5 i 0.7 (16) - modal 2 1 APICAL. PALISSADE DENDRITES Number per cell (Golei) 50 i 7 (61 29 t 9 (9) Sagittal distance (semithin) 3.1 ? 0.4pm (61 4.4 2 0.3 pm (6) Transverse distance (semithin) 2.5 ? 0.3 p m (4) 2.9 2 0.3 pm (4) 9% secondary apical branches 9 t 4 (51 % 26 2 12 (10) 9% (Golgi) Spine density apical (ultrathin) 14.5 t 1.6 per p m (41 14.4 f 4.1 per pm (31 middle (ultrathin) 9.7 f 3.1 per pm (3) 14.4 f 1.8 per pm (4) 13.6 f 3.6 per pm (4) basal (ultrathin) 5.9 % 1.4 per km (3) Spine number per dendrite (ultrathin) 2.000 f 540 ( 3 ) 2 800 f 400 (4) per cell (ultrathin) 100.000 i 14.300 (31 80.000 2 27.200 (41 Density of synaptic contacts apical (ultrathin) 10.1 f 1.7 per pm (41 10.4 t 4.0 per km (3) middle (ultrathin) 8.1 f 4.1 per pm ( 3 ) 10.7 i 3.0 per pm (4) basal (ultrathin) 4.4 t 1.5 per km ( 3 ) 9.5 t 2.7 per km (4) Number of synaptic contacts 2.000 t 440 (4) per dendrite (ultrathin) 1.500 i 645 (3) Der cell (ultrathin) 75.000 f 12.700 (3) 60.000 t 22.800 (4) PROXIMAL RECEPTIVE SURFACE Number of climbing fiber synaptic 123 t 36 ( 7 ) 130 i 23 (101 contacts (EM reconstr.) Number of contacting Purkinje 16 t 7 (10) axonal boutons (EM reeonstr.) 38 t 8 (7) AXONS Number of boutons (Golgi) 18 f 5 (12) 68 t 12 (10 230 2 71 km (121 Rostrocaudal extension (Gnlgil 130 t 80 pm (17) Rostrocaudal displacement (Golgi) 132 f 104 Km (15) 50 t- 35 pm (13) ‘The technique used is indicated in parentheses. ‘Numbers indicate animals for estimations based on semi- and ultrathin sections, and neurons for estimations based on Golgi impregnations.

Purkinje dendrites and Bergmann glial processes, separated by “septa” of transversely running parallel fibers (Fig. 6). Morphometrical analysis of sagittal electron microscopical sections revealed that in lobe C, each septum contains on average 10.000 parallel fiber profiles of on average 0.12 ,um diameter (Table 4), without significant apical-basal differences in diameter or numerical density (on average about 50 per pm height). In Golgi preparations, it was observed that these parallel fibers arise from granular cells with a similarly homogeneous morphology; each granule cell has generally 3 or 4 short dendrites of 10-20 pm length, and one axon arising most frequently from a dendrite (Fig. 9). In sagittal sections of lobe C,, about 950 septa of parallel fibers are present, which means a total number of about 9 x lo6 sectioned parallel fibers per section. Comparison of this number with the estimated number of about 7 x lo6 granule cells in lobe C,yields an estimation of the average length of parallel fibers in lobe C, of 9/7 times half of the circumference of this lobe, which means a length of about 1.5 mm (deeply) to 2 mm (superficially). Parallel fibers make synaptic contacts with the spines of the apical Purkinje dendrites (Fig. 6D, 8). The synaptic contacts are of the asymmetrical type, i.e., with pronounced postsynaptic densities and clear, round presynaptic vesicles (Figs. 7D, 8B). Incidentally, also a dense-core vesicle was observed in a parallel fiber bouton. The mean trace length of the parallel fiber synaptic contacts was 250 nm, which coincides with an average diameter of their synapticcontacts-disks of about 300 nm. Quantitative analysis of electron microscopic sections of lobe C, ,yielded a rough estimation of about 10 synaptic contacts with parallel fibers

167

per 1 pm Purkinje dendrite length (Table 3), which is remarkably smaller than the number of spines. Although this indeed might point to the existence of spines without synaptic contacts, similar to those observed by Harris and Stevens (’88)in the rat, we should be careful to suggest this since uncertainties in the estimation of the caliper factors of spines and synapses, might also be involved in this discrepancy. Since the precise caliper factor of spines with their variable shape and necks is more uncertain than that of synaptic contacts, we use synaptic densities and numbers for further calculations. A number of 10 synaptic contacts per mm would mean 2,000 per dendrite and thus about 60.000 per Purkinje cell. Since each Purkinje cell is passed by 300.000 parallel fibers (in 30 septa), this would mean that about 20% of these make contact with each Purkinje cell passed, and in turn that each parallel fiber would make on average one synaptic contact per about 5 Purkinje dendrites passed, which means about one per 15 pm or about 70 per parallel fiber of 1 mm length. However, it should be mentioned that they in addition make synaptic contacts with eurydendroid and stellate cells. In lobe C,, the morphology of synaptic contacts between parallel fibers and dendritic spines is similar to that in lobe C, as just described, but many quantitative differences are present with respect to density and number of contacts as well as with respect to the number and diameter of their parent parallel fibers. First, the septa of parallel fibers are thinner in lobe C, than in lobe C, and contain on average 3,500 parallel fiber profiles (Table 4).In the lower part of the molecular layer, both myelinated and unmyelinated parallel fibers occur (e.g., Fig. 8C), whereas superficially only unmyelinated parallel fibers are present. Dividing the molecular layer into three levels, it appears that the density of unmyelinated fibers (expressed as the number of parallel fibers per 10 pm septum height) decreases from 270 per 10 pm superficiallyvia 190 in the middle molecular layer to 80 in the deep part, whereas the density of myelinated parallel fibers increases from 0 superficiallyvia 2 in the middle part to 10 in the deep part of the molecular layer of lobe C,. More precisely, it seems that there exist at least three populations of parallel fibers in lobe C,, i.e. (1)a population of thin unmyelinated ones of about 0.13 pm diameter, with a decreasing density from superficial to deep, (2) a population of thick unmyelinated parallel fibers of about 0.35 pm diameter, with an increasing density from the middle to the deep level of the molecular layer, and (3) a population of still thicker myelinated parallel fibers (on average 0.45 pm) with a similar density distribution as the thick unmyelinated ones (Table 4).Most likely, the thick unmyelinated fibers are continuations of the myelinated ones after these have lost their myelin sheaths. In Golgi preparations, thin unmyelinated fibers in lobe C, could be observed to arise from small granule cells with a morphology similar to the granule cells of lobe C, (Fig. 9). Unfortunately, the origin of thick parallel fibers could not be established in our Golgi preparations. The fact that all ascending bundles of granule cell axons contain similar amounts of myelinated axons as the parallel fiber septa in the molecular layer suggests that they originate from (presumably large) granule cells within lobe C,. The larger average size of granule cells in lobe C, compared with lobe C, is in line with this suggestion. In sagittal sections of lobe C, about 1,400 septa of parallel fibers occur, each with about 3,500 parallel fiber profiles, which means a total of about 5.106profiles in one sagittal

,parallel

fiber septa,

Figure 6


Fig. 7. Light and electron microscopic details of Bergmann glial cells and their processes in the molecular layer of lobe C, and C,. A. Photograph B. drawing of Golgi-impregnated examples in lobe C , . C. Low magnification electron micrograph of two deimpregnated Bergmann glial processes in the molecular layer of lobe C , . D. High magnification of a deimpregnated Bergmann glial process bordering on an unimpregnated Purkinje dendrite (PI in a section neighbouring Figure 7C. E. Electron micrograph of an unimpregnated Bergmann glial cell body and its proximal processes in lobe C,. Magnifications: A: X500; B: ~ 4 5 0 ;C: X4000; D: X15000; E: X5000.

Fig. 6. The palisade pattern of the mormyrid corpus cerebelli as observed in the electron microscope. A. Sagittal section B. tangential section of the molecular layer of lobe C,, explained in C. D. Detail of a transversely sectioned dendrite in a tangential section of lobe C,, showing a spine making contact with a bouton originating from a parallel fiber (arrow). E. Sagittal section F. tangential section of the subpial region of the molecular layer of lobe C,, showing the endfeet of Bergmann glial processes covering the distal tips of Purkinje dendrites (cf, Fig. 7B), and dark, elongated glial cells covering the parallel fiber septa. Magnifications: A: ~6000; B: ~7000; D: ~ 2 5 0 0 0 E ; and F: X 10500.

i!

169

J. M E E K AND R. NIEUWENHUYS

170

TABLE 4. Quantitative Aspects of Mormyrid Parallel Fibers LOBE C, Thin unmyelinated fibers Parameter Number of profiles per parallel fiber septum (= number of fibers passing a palisade dendrite) Number (or density) per bni height of parallel fiber septum Superficial Intermediate Deep Diameter (pm) Estimated average length (pm) Number of synaptic contacts with Purkinje cell per pm p a r d lel fiber length

9,700

1,200 (3)

53 +- l ( 3 ) 46 i 6 (3) 46 i 10 (3) 0.12 0.01 (3)

LOBE C , Unmyelinated fibers Thin

Thick

3,200 i 600 (4)

350 i 70 (4)

Myelinated fibers 80 2 6

1,500-2,000

27 i 6 (4) 16 i 2 (4) 5 2 2 (4) 0.15 2 0.01 (4) 700

2 i 0.5 (4) 3 i 1.0 (4) 0.36 i 0.04 (4) 700

0.25 i 0.05 (4) 1 0 l(4) 0.44 2 0.04 (4) 700

0.07

0.14

0.40

0.40

*

*

section of lobe C,. This is remarkably about twice as much (10 per bm) via intermediate (8 per pm) to deep levels of the as the estimated number of granule cells in lobe C, (see molecular layer (4per pm). above) and might either suggest additional sources for Superficially,the ratio of the number of synaptic contacts parallel fibers in lobe C, or the occurrence of parallel fiber to the number of parallel fiber profiles is 10/27 (per pm collaterals. In Golgi preparations, the latter were indeed height), which means that about 37% of the small parallel observed. Several thick, deeply located parallel fibers ap- fibers that pass a dendrite make a synaptic contact with it, pear t o have large numbers of short, thin collaterals with and, conversely, that each small parallel fiber makes one many boutons (Fig. 9), which probably account for the synaptic contact per about 3 dendrites passed, which coinrelatively high number of thin parallel fiber profiles in cides with one contact per 7.5 pm or about 100 per parallel sagittal ultrathin sections. The number of boutons of the fiber (of about 700 p m length). In the deep molecular layer, collateral “swarms” of thick parallel fibers may be as much the unknown number of profiles belonging to collaterals of as 50 per 50-pm length of the parallel fiber and are not only thick parallel fibers interferes with calculations of contacts restricted to the parallel fibre septum of their parent fiber, per parallel fiber from the ratio between contacts and fiber but may cross several neighbouring septa and sometimes profiles, which is at this level 4 per 9 per pm height, or 44%. even penetrate the layer of Purkinje cells (Fig. 9). It is Of the 9 parallel fiber profiles per pm, on average 1 is unknown whether the thick fibers continuously give off myelinated, 3 are unmyelinated and thick, whereas the these collaterals during their course through lobe C,, or remaining 5 are thin unmyelinated profiles. When we assume that the latter 5 all belong to collaterals, the ratio whether they are restricted to certain hot spots. The presence of the thick parallel fiber collaterals renders observed would mean 4 contacts per 4 thick parallel fibers, it impossible to calculate the length of parallel fibers in lobe which means 1contact per 2.5 pm and about 300 per thick C, from the ratio between parallel fiber profiles in sagittal parallel fiber along its total length in lobe C,, values not sections and the number of granule cells, as could be done unlikely compared with Golgi observations. Consequently, for lobe C,j.However, in transversely sectioned Golgi prepa- the molecular layer of lobe C, shows a very pronounced rations, many parallel fibers could be observed to traverse differentiation in a superficial and deep part, with excluthe entire width of the molecular layer of C,, which is about sively thin parallel fibers superficially and probably almost 700 pm. The high ratio between parallel fiber profiles and exclusively thick parallel fibers deeply, with rather distinct granule cells suggests that indeed presumably all parallel densities of synaptic contacts. In lobe C, such a differentiafibers in lobe C, are as long as the entire width of the tion is absent, whereas in this lobe the average number of molecular layer. It should be noted that not all parallel parallel fibers per septum is much higher, but the number fibers in lobe C, originate from a T-shape arborization of an of their synaptic contacts per pm length is lower. ascending granule cell axon: laterally located ascending Relations with stellate cells axons only change direction from “ascending” to “parallel” without branching (Fig. 19B). In addition, most T-shape In the molecular layer, stellate cells are dispersed among granule cell axon branchings are asymmetrical, the ipsilat- the palisade dendrites of the Purkinje cells and their axons era1 branch being smaller than the contralateral one (Fig. presumably make synaptic contacts with these elements, 19B). Only in the midline region of lobe C, symmetrical just as has been shown in a variety of other vertebrates. T-shapebifurcations occur. It is presently uncertain whether “asymmetrical” parallel fibers occur in lobe C, as well. Quantitative analysis of ultrathin sections of lobe C, has 8. Electron micrographs of deimpregnated Purkinje dendrites yielded an estimation of 7 synaptic contacts between paral- in Fig. the molecular layer and their spiny contacts with parallel fiber lel fibers and dendritic spines per pm length of Purkinje boutons. A. Branching dendrite in the molecular layer of lobe C,. B. dendrites (Table 3). Just as observed in lobe C,, this density detail of a section neighbouring the one shown in A, with several is lower than the calculated spine density. About 7 synaptic deimpregnated spines that make synaptic contact with parallel fiber contacts per pm dendritic length would mean about 1,500 boutons. Arrow points to the connection between the central houton contacts per dendrite and about 75.000 per Purkinje cell, a and its parent thin parallel fiber. C. Part of a long palisade dendrite in number comparable to that calculated in lobe C,. The lobe C,. Notice the occurrence of thick, myelinated parallel fibers in this region. D. Proximal Purkinje dendrite in lobe C, giving rise to a number of synaptic contacts per pm dendrite length de- secondary smooth branch that traverses the region of densely packed creases, in correlation with the number of spines as well as cell bodies of Bergmann glial cells to reach the molecular layer. with the number of parallel fiber profiles, from superficial Magnifications: A: ~9000; B: ~36000; C: x 18000; D: x4500.

Figure 8


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However, in Gnathonemus no definite proof is presently available of such contacts. Mormyrid stellate cells appear to be very resistant to our Golgi-impregnations, and consequently we observed only 5 well-impregnated specimens, i.e., with both dendritic and axonal arborizations, all in lobe C, (Fig. 10). These had 4 to 5 thin dendrites without spines, with a less strict palisade pattern than Purkinje dendrites. Most likely, these dendrites receive synaptic contacts from parallel fibers, since all thin a-spiny dendrites observed in the molecular layer of lobe C, and lobe C, in normal electron microscopical material appear to do so. However, in such preparations stellate dendrites cannot be distinguished from dendrites of small eurydendroid cells, which are cerebellar projection cells located in the layer of Purkinje cells. Because of the low frequency of Golgi impregnation of stellate cells, we have presently no satisfactory Golgi-EM material available from these elements. The axons of four of the Golgi-impregnated stellate cells observed in lobe C, make small boutons in a region around the cell body, whereas one axon was deviating from this pattern, having several collaterals ascending to the superficial molecular layer (Fig. 10). Because of the absence of Golgi-EM material, we could not identify the boutons of stellate axons at the ultrastructural level. In normal EM material we equally could not recognize stellate axonal terminals with certainty, probably because of their relative low frequency and their small size, not deviating from parallel fiber boutons. Moreover, we did not observe contacts on Purkinje cells between spines, which could indicate a possible origin from stellate cells. Consequently, we are presently uncertain whether stellate axons terminate both on Purkinje dendrites and eurydendroid dendrites or only on one of these elements, and also whether stellate axons terminate preferentially at the lower part of these elements, as coud be expected from the view of efficacy of inhibition. Our Golgi preparations show several cells with boutons at higher level, so these occur as well. However, stellate cells are preferentially located at the lower molecular layer or even between the somata of Purkinje cells, and so the quantity of stellate axonal terminals in the deep molecular layer may be substantially higher than superficially. With respect to the synaptic input of stellate cells, a remarkable difference was observed between deeply located stellate cell bodies in lobe C, and C,: stellate cells located in the ganglionic layer of lobe C, appear to be frequently contacted by Purkinje axonal terminals (Fig. lOE), whereas such contacts seem to be entirely absent in lobe C,. The ultrastructural characteristics of Purkinje axonal terminals are described below. It should be noted that basket cells are absent in teleostean cerebella, including the gigantocerebellum of mormyrids. Accordingly, stellate- or basketlike contacts on the cell bodies and/or proximal dendrites of Purkinje cells were not observed in lobe C, and C,.

average 10 palisade dendrites, leading to the ultimate number of on average 50-60 apical palisade dendrites per Purkinje cell in lobe C, (Table 3). Although variable, the branching pattern of proximal dendrites in lobe C, is always regular in the sense that “crossing overs” rarely occur, i.e., proximal dendrites of the same cell crossing each other in the sagittal plane (Fig. 11).This means that there exists a certain regularity or even hierarchy in the relations between dendritic tufts and their parent cell body, since palisade dendrites above the cell body have the shortest dendritic connection with the parent cell body, whereas more peripherally, either caudally or rostrally located dendrites, have on average connections of increasing length with their parent cell body (Fig. 11). In lobe C,, the most frequently encountered type of Purkinje cell in Golgi preparations has only one short main dendritic trunk, or primary dendrite, from which 3 to 7 secondary branches arise, but cells with 2 or 3 primary dendrites were also observed (Fig. 16). The general appearance of the proximal dendritic tree is less regular in lobe C compared with lobe C,, since “crossing overs” of proximal dendrites frequently occur, disturbing the hierarchical pattern of increasing distances between dendritic tufts and cell body from centrally to more rostrally or caudally located dendritic tufts, as observed in lobe C,. The lower degree of regularity of the dendritic tree of Purkinje cells in lobe C, compared to lobe C, is also indicated by the frequent. location of the palisade dendrites into more than one sagittal plane, and by the more frequent occurrence of branching palisade dendrites, as described above. The best impregnated Purkinje cells in lobe C, had on average a number of 30 palisade dendrites with a maximum of 48 (Table 3). In the electron microscope, the ultrastructural characteristics of mormyrid Purkinje cells as described earlier (Kaiserman-Abramof and Palay, ’69; Nieuwenhuys et al., ’74) were confirmed by analysis of Golgi-impregnated and goldtoned neurons. Characteristic features of Purkinje cells include the occurrence of many cisterns of smooth endoplasmatic reticulum, the occurrence of mitochondria with a dark matrix and a dark cytoplasmatic matrix in general. They contain a nucleus with two nucleoli. All properties described deviate substantially from eurydendroid neurons and their proximal dendrites in the ganglionic layer, and consequently readily allow for identification of Purkinje cells in the mormyrid cerebellum, also when these are not Golgi-impregnated or labelled with another marker (Fig. 12). In addition, Purkinje cells appear to have deviating synaptic contacts compared with eurydendroid neurons, and in lobe C, a substantially lower density of contacts as well, criteria that also help to distinguish Purkinje cells from eurydendroid cells in unimpregnated electron microscopical material. Details of the synaptic contacts of Purkinje cells are given below, whereas the synaptic contacts of

The proximal Purkinje dendrites in the ganglionic layer In Golgi preparations, the somata of Purkinje cells have an average diameter of 26 pm in lobe C, and of 22 pm in lobe C, (Table 3). In lobe C,, they mostly give rise to 2 main dendritic trunks or primary dendrites, but other numbers, varying from 1 to 5, have also been observed. By way of a rather variable branching pattern (Figs. 4B, ll),the proximal dendrites give either directly or via secondary and tertiary branches, rise to 5 or 6 dendritic tufts with on

Fig. 9. Light microscopic pictures of Golgi-impregnated parallel fibers in lobe C , . A. Drawing of granule cells and their ascending axons (left below) and of the variability of parallel fibers encountered in sagittal sections. Notice the occurrence of thick parallel fibers with many thin, varicose collaterals in the deeper part of the molecular layer. B. Photograph of a through-focus video display of two thick parallel fibers and their collateral swarm in lobe C,, obtained with the method of Van der Linden et al. (’89).Magnifications: A: x 725; B: x2600.


r molecular layer

ganglionic layer

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with a pleiomorphic vesicular content (Figs. 12B, 13C-E). Remarkably, these profiles make desmosomelike contacts, both with the larger presynaptic “climbing” fiber boutons and with the necks of the postsynaptic thorns (Fig. 13C-E). Synaptic contacts with “climbing” fibers It is uncertain whether these profiles represent a second The somata and proximal dendrites of Purkinje cells, and type of “climbing” fiber boutons, or another type of neural sometimes also the initial parts of the axons, are contacted (or glial) element penetrating the climbing fiber glomeruli. by two types of terminals (Fig. 12A,B), one arising from By means of analysis and reconstruction of serial ulPurkinje cell axons (see below), and the other most proba- trathin sections of Golgi-impregnated as well as nonimpregbly representing terminals of olivocerebellar “climbing” nated Purkinje cells (Fig. 17), quantitative aspects of the fibers. Before describing the properties and distribution of distribution of “climbing” fiber terminals were studied. the latter type of terminals, indicated as “climbing” fiber “Climbing” fiber terminal clusters appear to be preferenterminals, two precautions should be made. First, these tially located on the most proximal part of primary denterminals do not, seem to climb into the molecular layer, but drites, sometimes partly including the neighbouring surare only located at the proximal parts of dendrites and/or on face of the cell body or even of the initial part of the axon the soma in the ganglionic layer; hence the placing of (Fig. 17). Each proximal dendrite has at least one cluster of “climbing” in quotation marks. Second, there is no definite “climbing” fiber contacts, and consequently they always proof of the origin of the terminals to be described from the seem to occupy a very strategic position in the signal inferior olive. However, an olivocerebellar projection to lobe pathway between the apical dendritic tufts and the axon C, and C, has been clearly demonstrated (Meek et al., hillock. Sometimes a cluster of “climbing” fibers is present ’86a,b), and other candidates for synaptic terminals from at a more distal location of a basal dendrite (Fig. 17). In lobe such axons have not been observed in the electron micro- C,, Purkinje cells make on average 130 synaptic contacts scope. Moreover, the ultrastructural characteristics and the with “climbing” fibers, divided over 2 to 4 clusters (Table 3; synaptic contacts with short, blunt spines or thorns are so Fig. 17). similar to the characteristics of olivocerebellar “climbing” In lobe C,, the organization of “climbing” fiber contacts fibers in other vertebrates (see Kaiserman-Abramof and on the surface of Purkinje cells shows several differences Palay, ’69) that it is presently most conceivable to assume compared with lobe C,, although their general preference that the mormyrid “climbing” fibers are homologous to the for a proximal location is similar. However, in lobe C, the olivocerebellar climbing fibers in other vertebrates. Conse- postsynaptic thorns are sometimes less well developed than quently, we followed the identification of mormyrid in lobe C, and may even be absent (Fig. 14); small profiles “climbing” fiber terminals as presented by Kaiserman- with pleiomorphic vesicles and desmosomelike contacts Abramof and Palay (’69), who described their ultrastruc- were not observed, and more frequently small dendrites t u r d features in detail. Attempts to identify the mormyrid without “climbing” fibers, as well as dendrites with two “climbing” fibers in lobe C, and C, as olivocerebellar ones clusters were observed (Fig. 18). This correlates with the by means of HRP injections into the inferior olive (brain- less regular arborization pattern of proximal Purkinje stem) and subsequent LM and EM investigation of antero- dendrites in lobe C,, and with the frequent occurrence of gradely labeled terminals failed until now, since we could monodendritic Purkinje cells. In lobe C,, Purkinje cells have not achieve anterogradely labeled terminals, probably be- on average 125 “climbing” fiber contacts, divided over 1-3 cause of the small diameter of olivocerebellar axons and the clusters (Table 3; Fig. 18). large degree of collateralization in the cerebellum (Meek et Our light microscopic observations on (presumed) al., ’86a,b). “climbing” fibers are scarce, since they appear to be very In lobe C,, the proximal dendrites, cell body and initial resistant to Golgi-impregnation or to anterograde tracing part of the axon of Purkinje cells may have groups of spines with HRP. However, in lobe C, we observed still a few and thorns, as is both visible in Golgi preparations (Fig. 4B) Golgi-impregnated axons most likely representing olivocerand electron microscopical sections (Fig. 13). Most of these ebellar “climbing” fibers judging from their properties. The thorns make synaptic contacts of the asymmetrical type best impregnated example is shown in Figure 13A. The and have very pronounced postsynaptic thickenings, fre- characteristics of these Golgi-impregnated “climbing” fiquently with additional subsynaptic dense material. How- bers are as follows. They arise from thin axons in the fiber ever, several thorns without synaptic contacts, but still layer situated between the layer of Purkinje cells and the containing a distal region with dense material, were also granular layer and give off a rich plexus of arborizations in observed. The presynaptic elements of these proximal spines are in lobe C, predominantly rather large boutons, although smaller profiles occur as well, probably partly representing Fig. 10. Light and electron microscopic pictures of Golgi-impregtips of larger boutons. In material treated for the Golgi-EM procedure, such boutons generally contain a low density of nated stellate cells in lobe C, and C,. A. Drawing of five impregnated large, round, clear vesicles of about 50-nm diameter, and examples in lobe C,. Arrows point to their axons. B. Photograph of a through-focus video display of the second cell shown in A, obtained by some dense-core vesicles (Figs. 12B, 13B). However, in means of the method of Van der Linden et al. (’89). Arrows point to the perfused, normal EM material the density of both clear and axon, C . Low magnification electron micrograph of an unimpregnated dense-core vesicles is higher. The presynaptic boutons, stellate cell and one of its dendrites in the lower molecular layer of lobe presumed to represent “climbing” fiber terminals, occur in C , . Notice the high density of nonspiny synaptic contacts on the surface groups, ensheathed by several laminae of glial tissue, thus of this dendrite. D. High magnification of part of the dendrite shown in C. E. Cell body and proximal dendrite of a stellate cell in lobe C,, located establishing a kind of glomeruli with clusters of thorns. in the boundary region between the molecular and the ganglionic layer. vesicle-containing boutons deApart from the large, Notice the occurrence of synaptic contacts with boutons of Purkinje scribed above, another type of profile was encountered in axons (arrows). Magnifications: A: ~ 6 0 0 ;B: ~1350;C: ~ 6 0 0 0 ;D: the “climbing” fiber glomeruli of lobe C,, i.e., small profiles X 12000; E: ~ 5 0 0 0 .

eurydendroid neurons are described elsewhere (Meek and Nieuwenhuys, in preparation).


Figure 10

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176

a

formation of presynaptic boutons in the same “climbing” fiber glomerulus, eventually might represent collaterals from the same parent axon. In lobe C,, Golgi-impregnated elements that could represent “climbing” fibres were not observed.

Purkinje cell axons and their terminals Although the axons of Purkinje cells appear to be much more resistant to Golgi impregnation than their dendrites, we still could study a substantial number of well-impregnated axons (Table 3; Figs. 4C, 15, 16). In lobe C,, the axon originates from the cell body or a proximal dendrite, becomes very thin after a distance of about 25 km (diameter decrease to about 0.2 km), and starts to ramify after an average distance of 40 pm from the axon hillock, giving rise to a rather large axonal terminal arbor with on average about 70 boutons of 1-2 pm width and 2-6 pm length in the best impregnated examples in lobe C, (Fig. 16). The Purkinje axonal terminal arbors in lobe C, have an average rostrocaudal extent of about 230 pm (100-350) but are in the transverse plane restricted to 50 to maximally 150 km (1-3 neighbouring sagittal sections), indicating that the d axonal terminal fields of Purkinje cells, although less strictly than their dendritic trees, are located in a sagittal zone as well. In lobe C,, the central point of the axonal terminal field is only slightly displaced rostrally or caudally compared to the cell body, which means that on average the axonal termination field is located right underneath the e dendritic tree of each Purkinje cell. Axons or axonal collaterals either penetrating the molecular layer or the granular layer were not observed, so the terminals appear to be restricted to the ganglionic layer, where dendrites and cell bodies of Purkinje cells and eurydendroid cells are the predominating postsynaptic elements. f In lobe C,, the axons of Purkinje cells show two main quantitative differences in Golgi-impregnated material compared with lobe C,. First, the initial part of the axon, i.e., the axon between the axon hillock and the first branching point, is much longer in C, (on average about 130 pm, varying from 50-500) (Table 31, which may lead to a substantial displacement (either rostrally or caudally) of the axonal terminal field compared to the dendritic tree of the same Purkinje cell (Figs. 4A, 151, although some axons have a similar distribution of terminals as in C,, i.e., around the cell body underneath the cell‘s own dendritic tree. Second, the axonal terminal arbor in the ganglionic layer is less extensive in lobe C, compared to lobe C, and makes on average only 20 boutons in the ganglionic layer (varying from 12-35), which are, however, substantially larger in Fig. 11. Examples of Golgi-impregnated cell bodies and proximal diameter than those in lobe C, (1-3 pm width and 2-10 m dendritic trees of Purkinje cells in lobe C,, to show their variability, which is remarkably large compared with the regularity of their distal length). Although one might argue that the low number of dendritic trees (cf. Fig. 5). The cell body may give rise to two primary boutons observed in lobe C, could be the result of incomdendrites (a and b),as well as to three ( c ) , four (d,ej,or even five (0, plete impregnation, our EM observations do not suggest whereas rather i r r e u l a r shapes also occur (g,h).Magnification: X 250. that this is a major factor involved (see below). Just like in lobe C,, the terminal fields of Purkinje axons in lobe C, are located in a sagittal zone, restricted to the ganglionic layer. the layer of ganglion cells, with several short terminal Axonal branches outside the ganglionic layer were not branches. Sometimes the axon has a “whorl”-like configu- observed, which is in line with our conclusion from HRP ration (Fig. 13A). The terminal branches were only ob- studies that mormyrid Purkinje cells are interneurons served in the layer of Purkinje cells and never ascended into (Meek et al., ’86a,b). the molecular layer. Sometimes the short, varicose collaterIn the electron microscope, the somata, proximal denals of different axons have a very close apposition, suggest- drites, and initial parts of the axons of Purkinje cells appear ing that they participate in the same “climbing” fiber to be contacted, apart from the “climbing” fibers, by a glomerulus or cluster. However, we cannot exclude the second type of bouton, with an electron-dense matrix, dark possibility that these different axons participating in the mitochondria, and a high density of small flattened vesicles,


Fig. 12. Electron micrographs of the cell body and proximal dendrite and axon of an unimpregnated Purkinje cell and its synaptic contacts. A. Low magnification, showing the nucleus, nucleolus and various cytoplasmic constituents of the cell body, proximal dendrite (left above) and initial part of the axon (left below), and the low density of synaptic contacts. Arrowheads point to contacts with Purkinje

177

axonal boutons and asterisks indicate “climbing” fiber boutons. B. Detail of a neighbouring section, showing a high magnification of a Purlunje axond bouton (left) and a “climbing” fiber bouton (right), which make synaptic contact with the cell shown in A at the origin of the dendrite. Magnifications: A X6000; B: X27000.

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Fig. 13. Light and electron microscopical details of “climbing” fibers and their synaptic contacts with Purkinje cells in lobe C,. A. Drawing of a Golgi-impregnated presumed “climbing” fiber in lobe C, as present in three consecutive 50-pm sagittal serial sections. B. Electron micrograph of a large “climbing” fiber bouton which makes synaptic contacts with a number of thorns of a Purkinje cell in lobe C . .

J. MEEK AND R. NIEUWENHUY:,

C-E. Details of the “shunting” elements present in “climbing” fiber glomeruli in lobe C,, which make desmosomelike junctions with both the spine necks and the “climbing” fiber boutons (C,E), and which contain small pleiomorphic vesicles and mitochondria (D,E).Magnifications: A. ~ 5 0 0B: ; ~24000;C: x 19000; D,E: ~ 3 2 0 0 0 .


Fig. 14. Examples of climbing fiber synaptic contacts in lobe C,. These may contact well-developed spines (A),but also thorns without a neck (B)or even the smooth surface of Purkinje dendrites (C).Notice the presence of a marked subsynaptic density at the sites of poorly developed thorns in B and C. Magnifications: A: x 16000;B: ~ 2 4 0 0 0 C: ; ~9000.

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180

a

9-

b

C

Fig. 15. Drawings of Golgi-impregnated axons of Purkinje cells in lobe C,. The axonal terminals may be located underneath the dendritic tree (a),but may also show a considerable rostra1 or caudal displacement (b-d),which may even be larger than depicted here (cf. Fig. 4A).

The axon generally originates from the soma, but sometimes from a proximal dendrite (c). Several close appositions between the dendrites and axonal boutons of the same neuron strongly suggest the presence of autapses (a). Magnification: X500.

which make distinct symmetrical contacts (Fig. 12B). Similar terminals make contacts with the receptive surface of eurydendroid cells. Their dense matrix and dark mitochondria as well as their size, shape, and distribution clearly suggest that these boutons represent the terminals of Purkinje axons. This was corroborated for two Golgiimpregnated and subsequently deimpregnated axons, which were followed in serial sections up to the level where they indeed started to make terminals of the type described above. The same reconstructions used for analysis of the distribution of “climbing” fibers on the receptive surface of Purkinje cells were used to study quantitative properties of Purkinje axonal boutons. In lobe C,, each Purkinje cell receives contacts from about 40 randomly distributed Purkinje axonal boutons (Table 3; Fig. 18). The symmetrical

contact zones have quite variable lengths and patterns in ultrathin sections, suggesting a very irregular shape, substantially deviating from a disklike one. Consequently, the number of synaptic contacts could not be estimated by morphometric methods, and the only parameter that could be determined was the number of terminals contacting the Purkinje cells, being about 40 in lobe C,, as stated above. Purkinje axonal terminals do not show preference for any site of the receptive surface of Purkinje cell bodies or proximal dendritic trees, except perhaps for the region around the clusters of “climbing” fiber synaptic contacts, where Purkinje axonal synaptic contacts were frequently present. Judging from the Golgi preparations, most Purkinje axonal boutons contacting one Purkinje cell probably arise from Purkinje cells located rostrally or caudally from that Purkinje cell. However, several axonal boutons of

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a

U

lobe C3 Fig. 16. Drawings of Golgi-impregnated axons of Purkinje cells in lobe C,. The number of boutons is much higher than in lobe C, (cf. Fig. 151, but the rostra1 or caudal displacement is on average smaller. Notice that most neurons in lobe C, give rise to only one primary dendrite (b-d), although higher numbers occur as well (a).Magnification: x 500.

Purkinje cells showed a very close apposition with the proximal dendrites of their own parent cells, suggesting the occurrence of autapses as well, i.e., synapses between the axon and dendrites of the same cell (Fig. 16). This enhances the impression of a completely random organization of Purkinje axonal synaptic contacts on the Purkinje cell dendritic surface, since they do neither discriminate between different compartments of Purkinje cells, nor between their parent Purkinje cell and neighbouring ones. This randomness may be extended to the contacts with eurydendroid neurons, since these also appear to have a similar density of Purkinje axonal synaptic contacts on their proximal dendritic and somatic surface as Purkinje cells, with a similar random distribution, to be reported in a subsequent study. In lobe C,, reconstruction of ultrathin serial sections confirmed the differences with lobe C, as observed already in Golgi preparations, i.e., Purkinje cells are contacted by fewer Purkinje axonal boutons (ranging from 5 to 30, on

average about 151, which are, however, in general substantially larger than in lobe C, (Fig. 17). The latter apparently partly compensates for the lower number and might result in a comparable number of synaptic contact sites or percent of receptive surface occupied by Purkinje axonal synapses on the surface of Purkinje cells in lobe C, and lobe C,. Similar to lobe C,, several very suggestive examples of autapses were observed in lobe C, (Fig. 15A),although such contacts are not possible for the most “displaced” axonal terminal fields. Apart from Purkinje cells, Purkinje axons terminate on eurydendroid cells, which probably accounts for the differences observed between the numbers of boutons per axon and per Purkinje cell surface. Details on Purkinje axonal contacts with eurydendroid cells in lobe C, and C, will be reported in a subsequent study. In addition, deeply located stellate cells are also a target for Purkinje axons, at least in lobe C, (see above). In lobe C, such contacts were absent or at least very scarce, which correlates with the smaller

b

d Figure 17


Fig. 18. Serial section reconstruction of the synaptic contact distribution on the receptive surface of the cell body and proximal dendrites of some Purkinje cells in lobe C,. The encoding is as indicated for Figure 17. Notice the higher density of contacting Purkinje axonal boutons and

difference between the number of Purkinje axon terminals per axon and per receptive surface of a Purkinje cell in lobe C, [18(?5)-16(?7) = 2(+9)] than in lobe C, [68(?12)38(?8) = 30(*14)1.

Fig. 17. Serial section reconstruction of the synaptic contact distribution on the receptive surface of the cell body and proximal dendrites of some Purkinje cells in lobe C,. Dots indicate the position and density of synaptic contacts with “climbing” fibers, and larger profiles indicate the shape and position of Purkinje axonal boutons that make synaptic contact with the cells reconstructed. Black dots and boutons are located in front of the cell, whereas open dots and stippled boutons lie behind the cell. Notice that “climbing” fibers terminate on proximally located clusters of thorns, and that all dendrites receive some “climbing” fiber input. When the axon originates within a “climbing” fiber glomerulus, it may receive some “climbing” fiber synaptic contacts as well (b).Purkinje axonal boutons are more randomly distributed, and may also terminate on the initial part of the axon (d).Magnification: x 1100.

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their smaller size compared with lobe C, (fig. 171, and the presence of dendrites with at least two (b) or three ( c ) clusters of “climbing” fiber contacts, as well as without “climbing” fiber contacts (d).Magnification: X 1100.

DISCUSSION Several previous studies have paid attention to the remarkable regularity of the molecular layer of the mormyrid cerebellum and have presented detailed qualitative information about the morphology, cytology, and synaptology of mormyrid Purkinje cells and their palisade dendrites, using both light and electron microscopical techniques (Nieuwenhuys and Nicholson, ’69a,b; Kaiserman-Abramof and Palay, ’69; Nieuwenhuys et al., ’74). With respect to qualitative aspects, our observations are completely in line with these descriptions, to which the reader is referred for all kinds of histological details which are not discussed here. The main purpose of the present study is to focus on the spatial organization and quantitative aspects of the morphological and synaptic properties of mormyrid Purkinje cells, with the intention to detect features that may be specifically correlated with the characteristic dendritic palisade pattern, and thus might indicate


184 its possible functional significance. For this purpose, the discussion section first focuses on the organization of the Purkinje cell input, as provided by parallel fibers, climbing fibers, and Purkinje axons, comparing findings in lobe C, and C, with observations in teleosts without a palisade pattern, as well as with the well-known organization of mammalian Purkinje cells. Second, an attempt is made to evaluate the possible functional significance of the dendritic palisade pattern and correlated morphological and synaptic properties of mormyrid Purkinje cells for their integrative properties.

The mossy fiber-granule cell parallel fiber pathway Since the palisade dendrites of mormyrid Purkinje cells are predominantly involved in the processing of parallel fiber input, it seems useful to compare the properties of the mormyrid mossy fiber-granule cell parallel fiber circuit with that of other vertebrates. The following may be noticed in this respect. The length and diameter as well as the number of synaptic contacts of parallel fibers in mormyrids seem to be significantly smaller than in mammals. The parallel fiber length in lobe C, is about 700 pm; that in lobe C, is 1.5 to 2 mm according to our estimations. Since lobe C, is a single sheet of cerebellar tissue, all parallel fibers are probably of equal length and are moreover coextensive in the transverse plane. In lobe C, the variability may be larger in this respect. In mammals, parallel fibers are probably longer: previous estimation ranged for rats from 0.9 to 4.7 mm (Ito, '841, whereas the most recent results indicate a length of even 5 mm (Harvey and Napper, '88). In other mammals, estimations are in the same range (Ito, '84). The diameter of mormyrid parallel fibers is in lobe C , on average 0.12 pm, which is substantially smaller than in mammals (about 0.2 pm; Ito, '841, although parallel fibers of 0.1 pm are within the range of mammalian parallel fibers (Palay and ChanPalay, '74). The same holds for the large caliber parallel fibers in lobe C, of 0.35-0.45 pm diameter, since diameters up to 0.35 pm have been reported for cats (Palkovits et al., '71). Equally, an increasing diameter gradient from superficial to deep, as present in lobe C,, has been reported for mammals (Ito, '841, although this difference in mammals seems to be less pronounced than in lobe C,. Although the presence of thick, myelinated parallel fibers has also been described for mammals (see Ito, '841, such a rich plexus of rather long collaterals as reported for lobe C,, has to our knowledge not been reported before and consequently might be considered as a specialization correlated with the mormyrid palisade pattern. However, in lobe C, such axons do not occur, whereas parallel fibers equally do not show any gradient in diameter or density from superficial to deep, but Purkinje cells still have a clear palisade pattern in lobe

c,.

In lobe C,, about 175.000 parallel fibers, and in lobe C, about 300.000 parallel fibers traverse the dendritic tree of a single Purkinje cell, which is substantially or only slightly lower, respectively, than the number of 400.000 calculated for cats (Palkovits et al., '71). In cats, Palkovits et al. ('71) calculated in addition that about 1per each 5 parallel fibers traversing a Purkinje dendritic tree makes a synaptic contact with that tree, which means about 80.000 parallel fiber contacts per Purkinje cell, and about 110 contacts per mm parallel fiber. These values are all in line with the present estimations of about 70.000 contacts per Purkinje

cell in lobe C, and C,, a ratio of 1 per 5 parallel fibers contacting a dendritic tree traversed in lobe C, (or 70 per mm parallel fiber) or 1 per 3 for small parallel fibers in lobe C, (or 140 per mm parallel fiber). The ratio and number for thick parallel fibers in lobe C, seem to be higher (1:l or 400 per mm length). However, several indications suggest that the number calculated by Palkovits for cats are too low: Brand et al. ('76) found much higher numbers of varicosities per mm length of parallel fibers in cats, whereas a recent calculation of Napper and Harvey ('88) yield an estimation of 175.000 synaptic contacts with parallel fibers per Purkinje cell for rats, a number much higher than the 80.000 calculated by Palkovits et al. ('71) for the larger Purkinje cells of cats. The shorter length and smaller diameter of mormyrid parallel fibers compared with those of mammals is correlated with a substantial smaller diameter of granule cells (about 4 pm in lobe C, and C, compared with 5 to 8 mm in mammals (Ito, '84), and a lower number of dendrites, 3 or 4 compared with 2 to 7 Ito, '8411, which in turn is correlated with a much higher numerical density in mormyrids (about 5.000.000 granule cells per mm3 in lobe C,, and about 12.000.000 per mm3 in lobe C, compared with about 2.000.000 per mm3 in rats (Harvey and Napper, '88)). However, the ratio between granule cells and Purkinje cells (about 800:l in lobe C, and 300:l in lobe C,) is in the same range as values presented for mammals, where estimations vary from 250 to 900 in rats and 600 to 1,600 in cats (Ito, '84). The recent estimation of Harvey and Napper ('88) for rats (250:l) yielded a value similar to that of C,. The smaller size of granule cells and their lower number of dendrites in mormyrids suggest that the integrative properties of mormyrid granule cells are restricted compared with mammals. This is probably due to the existence of the precerebellar nucleus lateralis valvulae, a major source of mossy fiber input to lobe C, and C, (Meek et al., '86a,b), as well as to the cerebellum of other teleosts (Finger, '83; Wulliman and Northcutt, '88, '89). Conceivably, this large preprocessing station for mossy fiber input, composed of adendritic cells with large club endings on their surface (personal observations), partly takes over the both convergent and divergent integrative function of granule cells (cf. Ito, '84). In summary, it may be concluded that several differences exist between the mormyrid and mammalian mossy fibergranule cell parallel fiber circuit, but that these are not very pronounced and rather point to different cerebellar dimensions than to specific correlations with the mormyrid palisade pattern. The only marked feature is the presence of thick, partly myelinated fibers with large numbers of collaterals in the deep molecular layer of lobe C,, but such fibers are absent in lobe C,, where still a marked palisade pattern is present. Unfortunately, for teleosts without a palisade pattern, no detailed data concerning the organization of the mossy fiber-granule cell parallel fiber pathway are available (cf. Paul, '82; Finger, '83).

The olivocerebellar climbing fiber system In mammals it is well known that climbing fibers, indicated as such since they "climb" into the dendritic tree of Purkinje cells (Ramon y Cajal, '11):(1)make asymmetrical synaptic contacts on spines or thorns of the so-called smooth or proximal parts of the dendritic trees (Palay and Chan-Palay, '74; Ito, '841, (2) originate exclusively from the inferior olive (Ito, '84), (3) have a one-to-one relation with Purkinje cells since each Purkinje cell is only contacted by a

PALISADE PATTERN OF MORMYRID PURKINJE CELLS single climbing fiber (Palay and Chan-Palay, ’74), and (4) follow the parasagittal zonal organization of Purkinje cells, since specific olivar subdivisions terminate in restricted parasagittal zones (Voogd and Bigare, ’80; Ito, ’84; Voogd, ’89). Similar properties, with the exception of a clear parasagittal organization, seem to be present in amphibians (Sotelo, ’76; Cochran and Hackett, ’77; Van der Linden and Ten Donkelaar, ’90) and reptiles (Hillman, ’69; Kunzle and Wiklund, ’82). In frogs, Purkinje cells make on average about 300 synaptic contacts with their climbing fibers (Llinas et al., ’691, whereas for other animals such numerical data are not available to our knowledge. In mormyrids, synaptic contacts resembling mammalian climbing fibers have been characterized as such only on the basis of their ultrastructural features (Kaiserman-Abramof and Palay, ’69). In the trout, similar contacts were also by Pouwels (’78b) identified as climbing fiber contacts. We followed this identification and agree with it, in spite of the absence of a definite identification by means of anterograde tracing from the inferior olive, since (1)the ultrastructural features of mormyrid and other teleostean climbing fibers are indeed quite unique and similar to that of mammals (Kaiserman-Abramof and Palay, ’69; Pouwels, ’78b), (2) there is indeed an olivocerebellar projection in several teleosts (Finger, ’78a, ’83; Pouwels, ’78a) as well as to both lobe C, and C, of mormyrids (Meek et al., ’86a,b), (3) teleostean Purkinje cells may show complex-spike responses (see Finger, ’83),which are in mammals specifically and exclusively evoked by climbing fiber input (Eccles et al., ’67; Ito, ’84), and (4) the present study did not show any other type of Purkinje cell input that might represent an olivocerebellar one. It should be noted that the mormyrid inferior olive is not very large (Meek et al., ’86a), but still projects to all parts of the large corpus (Bell et al., ’81;Meek et al., ’86a,b) and to the huge valvula (Finger et al., ’81), just as it does in other teleosts (Finger, ’78a, ’83; Wulliman and Northcutt, ’88, ’891, which suggests a very large degree of intracerebellar collateralization of this olivocerebellar input in mormyrids. Accepting the identification of mormyrid “climbing” fibers on the basis of the criteria just discussed, two interesting differences with mammalian and other vertebrate climbing fibers may be noticed, i.e., (1)they do not climb into the molecular layer, and (2) they do not seem to have a one-to-one relation with Purkinje cells. The nonclimbing nature of mormyrid “climbing” fibers, apparent from the distribution of synaptic contacts on reconstructed Purkinje cells and -also suggested by Golgi-impregnated examples in the present study, might at first sight represent a distribution that is correlated with the dendritic palisade pattern of mormyrids. However, in teleosts without a cerebellar palisade pattern a similar configuration with “climbing” fiber terminals restricted to the layer of Purkinje cells, seems to be present (Finger, ’78a, ’83; Pouwels, ’78a,b). During development, climbing fibers in other cerebella pass similar development stages, with only proximal climbing fiber contacts, before they really climb into the dendritic tree (Ramon y Cajal, ’11; Palay and Chan-Palay, ’74; Van der Linden and Ten Donkelaar, ’90). Consequently, t h e teleostean configuration, including the mormyrid one, most likely represents a “primitive,” simple configuration rather than an advanced specialization correlated with the specialized palisade pattern. The functional advantage of really climbing fibers compared with the proximal location observed in teleosts is unclear at present.

185

It should be noted that the location of climbing fibers in mormyrids, close to the axon hillock and always interrupting the pathway between parallel fiber input and the axon hillock, already seems very effective for a maximal influence on Purkinje cell output. In addition, the number of climbing fiber contacts per Purkinje cell does not seem to increase proportionally with their distribution: in the frog they make about 300 contacts per Purkinje cell (Llinas et al., ’69), a number only about twice as high as the average of 130 contacts calculated for mormyrids. In rats, where data concerning their number of contacts appear not to be available (cf. Ito, ’84), pictures of well-impregnated climbing fibers also do show not more than about 300 boutons per Purkinje cell (cf. Palay and Chan-Palay, ’74). Apart from their proximal location, the absence of a one-to-one relation as suggested by Golgi-impregnated material might be a second difference between mormyrid and mammalian climbing fibers. A definite validation of these observations has to be made by means of anterograde labeling of climbing fibers in mormyrids, and subsequent electron microscopical analysis, as, e.g., recently achieved for Xenopus by Van der Linden and Ten Donkelaar (’go), who found a similar configuration in developing cerebella of Xenopus. In developing mammalian cerebella, a one-to-one ratio between climbing fibers and Purkinje cells is equally not yet present (Ramon y Cajal, ’11; Palay and Chan-Palay, ’74; Ito, ’84). Conceivably, the suggested absence of a one-to-one relation between mormyrid climbing fibers and Purkinje cells is a second “primitive” and “simple” characteristic, correlated with their proximal location, but not with the advanced palisade pattern of mormyrid Purkinje cells. In lobe C,, the climbing fiber glomeruli contained, apart from climbing fiber boutons and Purkinje cell thorns and their synaptic specialization, a third element, containing small vesicles or reticular elements, which made desmosomelike contacts with both climbing fiber boutons and Purkinje cell thorns. Assuming that these contacts have a function in electric coupling, they probably establish some shunting mechanism. To our knowledge, similar elements involved in climbing fiber synaptic inputs have not been observed in other cerebella. Their origin in mormyrids is unknown at present. It should be noted that such elements were not encountered in lobe C, and that consequently also this particular aspect of mormyrid Purkinje cells is not strictly correlated with the palisade pattern. A prominent feature of mammalian climbing fibers is their sagittal zonal organization, which follows the parasagittal zonation of Purkinje cells (Voogd and Bigare, ’80; Ito, ’84; Voogd, ’89). However, the distribution of immunoreactivity to Zebrin 11, a monoclonal antibody against Purkinje cells, shows that a parasagittal organization of Purkinje cells is absent in teleosts (Brochu et al., ’go), including mormyrids (personal observations). Most likely, the cerebellar corpus of mormyrids should be considered as a single sagittal zone of about 700 to 2,000 pm width, rostrocaudally folded into several folia or lobes (C, to CJ, but not further differentiated into the transverse plane. In accordance, mormyrid climbing fiber projections do equally not show a sagittal zonation, but only rostrocaudal differences (Meek et al., ’86a,b),just as in other teleosts (Finger, ’83).

Synaptic contacts of Purkinje cell axons Mormyrids lack deep cerebellar nuclei, since they have instead eurydendroid projection cells (Nieuwenhuys and


186 Nicholson, 69b; Nieuwenhuys et al., ’74; Meek et al., ’86a,b), a property shared with other teleosts (Finger, ’78b, ’83;Murakami and Morita, ’87).Accordingly, Purkinje cells are interneurons with axonal terminals restricted to the ganglionic layer. Remarkably, this is not correlated with a larger number of terminals per axon: these numbers, as counted in Golgi preparations (on average about 20 in lobe C, and 70 in lobe C,) are lower than estimations of the number of recurrent Purkinje axonal collateral boutons in the cat (about 100 or more per Purkinje cell; Bishop and O’Donoghue, ’86; Bishop et al., ’87; Bishop, ’88). Although the numbers estimated on the basis of Golgi preparations may represent an underestimation, this does not seem to introduce a very serious effect in our material, since countings of numbers of terminals on the receptive surface of Purkinje cells are in line with these numbers. Consequently, the discrepancy between a tremendous number of input synapses and only a low number of output synapses, seems to be in mormyrids even much higher than in mammals, where Purkinje cells not only have more terminals in the ganglionic layer, but in addition terminate in deep cerebellar nuclei. This discrepancy is highest in lobe C,, where, remarkably, the palisade pattern seems to be more regular at the individual neuronal level than in lobe c3.

In contrast to climbing fiber contacts, which are clustered on restricted parts of specific neurons (i.e., proximal parts of Purkinje cells), Purkinje axons seem to distribute their synaptic contacts randomly over various parts of different targets, including dendrites, cell bodies, and sometimes also initial axonal parts of neighbouring Purkinje cells, eurydendroid cells, and possibly their own parent Purkinje cell (by means of autapses) as well as deeply located stellate cells. With respect to the latter, however, again a difference between lobe C, and lobe C, was observed, since Purkinje cell axonal contacts on deeply located stellate neurons were frequently observed in lobe C,, but only incidentally in lobe C,, where many deeply located stellate cells seem to receive no input from Purkinje axons at all. About the axonal contacts of these as well as more superficially located stellate cells, presently nothing is known. We only know that they are not specifically restricted to the deep molecular layer, since more superficially located axonal boutons were observed in Golgi preparations as well.

The palisade pattern As described by Nieuwenhuys (’67) and Nieuwenhuys and Nicholson (’69b), both lobe C, and lobe C, show a very regular palisade pattern in their molecular layer because of the characteristics of the spiny dendrites of mormyrid Purkinje cells. Nevertheless, the present study showed that individual Purkinje cells show a different degree of regularity in lobe C, and lobe C,. Those in lobe C, are the most regular ones, since the rostrocaudal hierarchy in dendritic branches (absence of crossing overs), the filling of the dendritic field, and the strict location in a sagittal plane is better in lobe C, than in lobe C,, whereas the number of branched palisade dendrites is lower in lobe C,. In spite of this regularity, the basal, proximal dendritic trees of Purkinje cells in lobe C , still show many variations in branching patterns and organization. This suggests that the regularity is organized at the level of the molecular layer, e.g., by specificinteractions leading to one-to-one relations between the mormyrid Bergmann glia palisade processes and the Purkinje cell dendrites. Strict rules in the branching pat-

tern of Purkinje cells determined by intrinsic factors within each Purkinje cell are apparently not involved. In the previous section, several characteristics of the afferent pathways to Purkinje cells are discussed, since they possibly might be correlated with the mormyrid palisade pattern. Remarkably, in particular in lobe C, with its most regular Purkinje cells, a number of specializations are observed, including apical-basal gradients in spine density, parallel fiber density, and parallel fiber diameters, shunting elements present in the climbing fiber glomeruli and displacements of axonal arborizations compared with the dendritic tree. Although all these specializations will have their own functional significance, which well might depend on or interact with that of the palisade pattern, they do not point to the basic functional significance of the presence ofa palisade pattern, since lobe C, shows that palisade dendrites are also present, although somewhat less well organized, in cerebellar parts without these specializations. Other aspects of the mormyrid cerebellar organization, observed both in lobe C, ad C,, such as the fact that Purkinje cells are interneurons, and that “climbing” fibers do not climb but occupy a position on proximally located clusters of spines, are equally not strictly and indissolubly correlated with a palisade pattern, since it was shown that similar configurations occur in teleosts without a palisade pattern. Consequently, the palisade pattern is more likely correlated with intrinsic properties in signal processing and integration of Purkinje cells than with extrinsic factors in the organization of Purkinje cell input and output. In this respect the following may be noticed. In general, the dendritic tree of Purkinje cells is considered to be composed of two distinct compartments: the spiny branchlets, with their numerous spines that contact parallel fibers, and the “smooth” or “proximal” part, i.e., the primary, secondary, and tertiary dendrites that do not bear the numerous spines contacting parallel fibers, but only a low density of thorns that contact climbing fibers (Palay and Chan-Palay, ’74; Ito, ’84). A major difference between mammalian and mormyrid Purkinje cells is that the former have a large “smooth” part, located in the molecular layer, with many terminal spiny branchlets, each of which is, however, relatively short and has relatively few spines compared with mormyrids, whereas mormyrid Purkinje cells have a relatively small “smooth” part, restricted in location to the ganglionic layer, with only few, but very large spiny (palisade) branchlets, each of which has an enormous number of spines. Thus comparison of mammals and mormyrids suggests an important difference in the balance between intradendritic and interdendritic integration of parallel fiber input, i.e., interactions within each spiny branchlet between spines, and interactions between different spiny branchlets on the “smooth” part of the Purkinje dendritic tree, respectively. In Mormyrids, each palisade dendrite has to integrate input from many spines, but the smooth part has only to integrate input from few dendrites, whereas in mammals the smooth part has to integrate input from many spiny branchlets, which each, however, integrate input from a relatively low number of spines. With respect to intradendritic signal interactions, i.e., with respect to the function of spines, several possibilities have been proposed on the basis of theoretical considerations and computer simulations. Although one of the functions of spines may simply be a mechanic one, i.e., allowing large numbers of dendrites and axons to make


187

efficient synaptic contacts without zig-zagging (Peters and electrically "passive" spines, spike generating ones are Kaiserman-Abramof, '70; Swindale, '81), most studies con- supposed to be a very effective, or even better, substrate to sider the spine neck as a locus of relatively high electrical regulate the weight and efficacy of synaptic contacts in resistance and/or chemical isolation between the synaptic space and time (Miller et al., '85; Perkel and Perkel, '85; contactb) located on the spine head and its parent dendrite. Segev and Rall, '88). Compared with direct axo-dendritic contacts, this would It is presently unknown which of the functions just result in the effect that a certain synaptic conductivity discussed, including a simple connective one, strong or change induced by a given degree of presynaptic activation, weak attenuations, weight regulation, andlor enhancement leads to a larger postsynaptic depolarization (or even pseudo- and amplification of spike generation, is the most likely for spikes; Wilson, '84). In turn, this would counteract the the spines on the palisade dendrite of mormyrid Purkinje (passive) current flow through the spiny synapse and the cells, since nothing is known about their chemical and spine neck toward the parent dendrite, thus attenuating electric properties, nor about the electrophysiology of the ultimate effect of synaptic input on the spike generation mormyrid Purkinje cells as a whole. However, a simple at the axon hillock of the postsynaptic cell (Jack et a1.,'75; connective function, as proposed by Peters and KaisermanKoch and Poggio, '83; Wilson, '84; Pongracz, '85). Abramof ('70) and Swindale ('811, is not very likely, since Depending on the passive and/or active electrical and/or coexisting eurydendroid neurons receive equal numbers of chemical properties ascribed to the cytoplasm and mem- parallel fiber synaptic contacts on their apical dendrites, brane of spines, the general mechanisms described above without spiny specializations (personal observations). Conmay lead to either strong or weak attenuation, or to sidering the very large number of spines on one palisade enhancement and multiplication of synaptic input. For, dendrite (2,000-3,000), it seems more reasonable to aswhen the electrical resistance of the spine neck is supposed sume an attenuating function instead of an enhancing one, to be very high, this would result in strong attenuation or since the latter would lead to a maximal response after binarization of synaptic input, since already a small change stimulation of ony a small population of spines, which in conductance would yield the maximal postsynaptic depo- would reduce or even abolish the specificity or tuning of larization possible in a spine (Koch and Poggio, '83). Purkinje cells for particular patterns of parallel fiber activConsequently, in such situations spines may be considered ity. However, until more is known about the electrical as constant current injectors (Llinas and Hillman, '69), properties of these neurons and their spines, this cannot be subserving linear integration of synaptic input (Llinas and concluded with certainty. It should be noted in this respect Hillman, '69; Diamond et al., '70). However, recent estima- that it is not necessary to postulate for all spines in the tions suggest that the spine neck resistance is in general not vertebrate nervous system a similar mechanism and funchigh enough to subserve such functions (Harris and Stevens, tion. As Gray ('82) pointed out, large differences exist in '88, '89). Weakly attenuating spines would be a very morphology, not only in shape (e.g., Peters and Kaisermaneffective instrument in determining the weight of synaptic Abramof, '70; Crick, '821, but also in the occurrence of the input (Chang, '52) and could regulate spatial and/or tempo- spine apparatus, which is well elaborated in mammalian ral aspects of synaptic efficacy, since small changes in the telencephalic spines, but absent or very weakly developed in morphology of the spine neck and/or head could yield large other regions (Gray, '82). Thus the possibility that, e.g., electrical and/or chemical effects. Consequently, spatial hippocampal spines are active, underlying their involvediversity of spine properties on a neuron could compensate ment in long-term potentation (Wickens, '88) does not or enhance the electrotonic distance effect of synapse exclude the possibility that mormyrid palisade spines might location (Rall, '74; Wilson, '84; Pongracz, '851, whereas be passive and subserve an attenuating function. With respect to interdendritic interactions, i.e., interactemporal changes, assuming that spine morphology is changeable under the influence of synaptic activity and tions between dendrites, Rall('62, '64, '70) has proposed a experience, as has been shown by many investigators (see, model for the electrotonic properties and coupling of branche.g., Crick, '821, make spines very suitable as a basis for ing dendrites that seems to be a good approximation for the plasticity during development, learning, and memory forma- integration of signals from different spiny branchlets by means of the smooth part of the dendritic tree of frogs as tion (Chang, '52; Rall, '74; Koch and Poggio, '83). The attenuating effects of spines postulated above all well as mammals (Pellionisz et al., '77; Pellionisz and start from passive electrical membrane properties, but it is Llinas, '77). However, it is doubtful whether similar mechanot unlikely that voltage-dependent channels occur in the nisms are involved in interdendritic interactions of membrane of spines (e.g., Wickens, '88). When active mormyrid Purkinje cells, since they have no dichotomous electrical properties are indeed present in the spine mem- arborization pattern with gradually thinning dendrites. brane, and spines thus not only would be able to evoke Instead, each palisade dendrite, already rather thin at its pseudo-spikes (Wilson, '84) but also real action potentials, base, seems to be "isolated" from the proximal dendrites by their morphology is very appropriate to enhance postsynap- a densely packed layer of Bergmann glia cells and their tic spike generation (Koch and Poggio, '83;Miller et al., '85; proximal processes, which represent a pronounced glial Perkel and Perkel, '85; Pongracz, '85; Segev and Rall, '88; boundary between the ganglionic and molecular layer. Wickens, '88). This is not only due to the high input Consequently, mormyrid palisade dendrites may be considresistance of the spine neck yielding large intraspinal ered as rather independent giant or super spines, each of postsynaptic potentials after relatively little changes in which processes separately the input from a distinct set of synaptic conductancy (Koch and Poggio, '83; Wilson, '84), parallel fibers. It would be interesting to know more about but also or even predominantly caused by the effect of the electrical properties of palisade dendrites to be able to chemical isolation of dendrite and spine head by the spine evaluate whether they indeed might subserve similar attenneck, which allows for high concentrations of various uating or enhancing functions as just discussed for spines. Whatever the electrophysiological properties of the palisubstances in the spine head, in particular calcium (Horwitz, '84; Harris and Stevens, '88, '89; Wickens, '88). Like sade dendrites and their spines may be precisely, it is


188 REGULARITY

INPUT

GRADIENTS

NUMBERS (per cell)

fibers

75,000

I

LO"laL15

. Purklnje axons

16

boutons

'clirnblng' fibers

130

contic~s

mossy fibers

A

B

(sagittal)

(transverse)

Fig. 19. Summarizing scheme of the morphological and synaptic organization of Purkinje cells and their input in lobe C,. A. Sagittal view showing the distribution of input, the regularity, the gradients in spineand parallel fiber density and the numbers of synaptic contacts observed or estimated in lobe C,. B. Transverse view showing the similarity in length of the parallel fibers contacting Purkinje cells in

lobe C,, but their variability in diameter (superficially: thin; deeply: thick) and site of origin: medially located granule cells give rise to symmetrical parallel fibers (a; i.e., a left and right branch of equal length), but more laterally located granule cells have asymmetrical (b) or even unidirectional (c) parallel fibers. Magnifications: A x 150; B: x75.

obvious that horizontal (i.e., rostrocaudal) or vertical (apicalbasal) patterns of parallel fiber input are very differently processed by mormyrid Purkinje cells, i.e., the former predominantly by means of interdendritic integration, but the latter by intradendritic integration. For instance, parallel fiber input restricted to a small horizontal plane would stimulate an equal, but relatively small number of spines on all dendrites of a mormyrid Purkinje cell, whereas parallel fiber input restricted to a small vertical plane (or beam) would result in the excitation of all spines of only a few dendrites, however. When similar numbers of parallel fibers and spines are involved, the former is conceivably more effective than the latter. In mammalian Purkinje cells both situations seem to be equally effective, since they lead to equal amounts of intra- and interdendritic interactions. Several more complicated examples, including different apical-basal and rostrocaudal parallel fiber activity gradients or waves, show a similar larger specificity of mormyrid Purkinje cells. Consequently, we suggest that mormyrid Purkinje cells are more sharply tuned to specific spatiotemporal patterns of parallel fiber input than those of other

vertebrates and that this is the main, and probably only, significance of the mormyrid cerebellar palisade pattern. Unfortunately, almost nothing is known about such specific spatio-temporal patterns of parallel fiber activity and their relation to external or internal sensory and/or motor signals, which means that it is unknown at present why parallel fibers in fact run parallel. The considerations discussed above suggest that mormyrids with their cerebellar regularity might be an optimal object to study the functional significance of the parallel fiber organization more closely. Consequently, we are presently trying to develop a theoretical model for this aspect on the basis of the structural organization of lobe C,, where the most regular organization of individual Purkinje cells was observed (Fig. 19),suggesting that the parallel fiber organization may be very refined and specific as well. Interestingly, in this lobe parallel fibers have some properties not present (or not observable) in other cerebella, i.e., all parallel fibers seem to be of equal length and are coextensive in the transverse direction. Consequently, within each diameter class, they only differ with respect to their origin more


/-

PRIMITIVE (example : perch)

189

MAMMALS (‘climbing fiber specialization’)

‘ \ ‘\

‘\

‘?a

MORMYRIDS (,parallel f i b e r specialization,) Fig. 20. Comparison of mammalian and mormyrid Purkinje cells. The “smooth,” proximal dendritic compartment contacted by climbing fibers has been drawn thickly, whereas the spiny, distal dendritic compartment contacted by parallel fibers has been drawn thinly (omitting the numerous spines). Starting from a primitive or basal configuration as observed in most fishes, one could state that in

medially or more laterally, which results in the presence of symmetrical parallel fibers (i.e., with two branches at the T-bifurcation of equal length) via asymmetrical parallel fibers to unidirectional parallel fibers, i.e., parallel fibers only running in one direciton, either from left to right or from right to left (Fig. 19B). In a separate work we explore the functional significance of this particular parallel fiber organization in lobe C, more closely (Meek, in preparation).

mammals and other vertebrates in particular the “smooth” compartment is enlarged and has been displaced to the molecular layer to increase, refine, and/or optimalize climbing fiber influence on Purkinje cells, whereas in mormyrids and related teleosts with a palisade pattern the spiny compartment is specialized to increase, refine, and/or optimalize the influence of parallel fiber input on Purkinje cells.

enlargement of the smooth dendritic part in mammals, compared with mormyrids, and the penetration of the molecular layer of this dendritic compartment in mammals and other vertebrates. It is discussed above that the mormyrid situation probably represents the more primitive climbing fiber configuration and that the mammalian organization of climbing fibers is the most specialized one, as can be seen in phylogeny as well as ontogeny (see above). Furthermore, Mormyrid versus mammalian Purkinje cells the mammalian configuration is probably not necessary to Comparison of mormyrid and mammalian Purkinje cells increase the number of synaptic contacts between Purkinje does not only suggest substantial differences in the process- cells and climbing fibers, since this increase is only twofold ing of parallel fiber input, but also in the interactions as discussed above. Such an increase could easily be realized between parallel fiber input and climbing fiber input. Both without enlargement of the proximal surface, whereas it types of input are received by separate compartments of the has been argued that the mormyrid configuration seems Purkinje cell dendritic tree: parallel fiber input by the spiny already very effective. Consequently, the major significance dendritic tufts, and climbing fibers by the “smooth,” more of the mammalian climbing fiber configuration is the proximal part of the dendritic tree, which therefore is not penetration of the smooth dendritic part with its climbing entirely smooth but bears a special type of spines or thorns fiber input into the molecular layer, thus reducing the t o receive this input (Palay and Chan-Palay, ’74; Ito, ’84; distance and possible glia barriers between climbing fiber present study). In this respect two major differences exist input and parallel fiber input. Probably, this allows for between mormyrid and mammalian Purkinje cells, i.e., the more subtle synaptic and possibly also nonsynaptic interac-


190 tions between climbing fiber input and parallel fiber input in mammals compared with mormyrids and other teleosts. Taking the differences between mormyrid and mammalian Purkinje cells together, the latter, with its one-to-one relation with climbing fibers and a reduced distance between climbing and parallel fibers, might be indicated as a “climbing fiber specialization,” whereas it has already been argued that we consider mormyrid Purkinje cells as a “parallel-fiber specialization.” Thus starting from a “basic” (or “primitive”) Purkinje cell, as present in lampreys, elasmobranchs, and most actinopterygians (e.g., Ariens Kappers, ’36;Larsell, ’67; Nieuwenhuys, ’67; Nicholson et al., ’691, the mormyrid configuration would represent a specialization to extract maximal information from parallel fiber input, whereas mammalian Purkinje cells seem to be specialized t o gain maximal effects of climbing fiber input on different aspects of parallel fiber input processing (Fig. 20). The present study suggests that electrophysiological analysis of mormyrid Purkinje cells might be very rewarding in analyzing several aspects of climbing fiber, and in particular parallel fiber processing by Purkinje cells, which are very difficult or impossible to analyze in mammals,

ACKNOWLEDGMENTS The authors thank Mrs. D. Elsevier for histological assistance, Mr. T. Hafmans for photographical assistance, and Mrs. M. van de Coevering for typing the manuscript.

LITERATURE CITED Abercrombie, M. (1946) Estimation of nuclear population from microtome sections. Anat. Rec. 94.239-247. Albers, F.J., J. Meek, and T.G.M. Hafmans (1990) Synaptic morphometry and synapse-to-neuron ratios in the superior colliculus of albino rats. J. Comp. Neurol. 291:220-230. Albers, F.J., J. Meek, and R. Nieuwenhuys (1988)Morphometric parameters of the superior colliculus of albino and pigmented rats. J. Comp. Neurol. 274:357-370. Ariens Kappers, C.U., G.C. Huber, and E.C. Crosby (1936) The Comparative Anatomy of the Nervous System of Vertebrates, Including Man, 2nd ed., 1967. New York Hafner. Bell, C.C. (1986) Electroreception in mormyrid fish: Central physiology. In Bullock and W. Heiligenberg (eds): Electroreception. New York: John Wiley & Sons, pp. 423452. Bell, C.C., and T. Szaho (1986) Electroreception in mormyrid fish: central anatomy. In T.H. Bullock and W. Heiligenberg (eds): Electroreception. NewYork: John Wiley & Sons, pp. 375421. Bell, C.C., T.E. Finger, and C.J. Russell (1981) Central connections of the posterior lateral line lobe in mormyrid fish. Exp. Brain Res. 42r9-22. Bishop, G.A. (1988)Quantitative analysis of the recurrent collaterals derived from Purkinje cells in zone X of the cat’s vermis. J. Comp. Neurol. 274: 17-31. Bishop, G.A., and D.L O’Donoghue (1986) Heterogeneity in the pattern of distribution of the axonal collaterals of Purkinje cells in zone b of the cat’s vermis: An intracellular HRP study. J. Comp. Neurol. 253r483499. Bishop, G.A., T.L. Blake, and D.L. O’Donoghue (1987) The distribution pattern of Purkinje cell axon collaterals: variations on a theme. In J.S. King (ed): New Concepts in Cerebellar Neurobiology. New York: Alan R. Liss, Inc., pp. 29-56. Blackstad, T.W. (197511 Electronmicroscopy of experimental axonal degeneration in photochemically modified Golgi preparations: A procedure for precise mapping of nervous connections. Brain Res. 95r191-210. Brand, S., A.-L. Dahl, and E. Mugnaini (1976)The length of parallel fibers in the cat cerebellar cortex. An experimental light- and electronmicroscopic study. Exp. Brain Kes. 26:39-58. Brochu, G., L. Maler, and R. Hawkes (1990) Zebrin 11:A polypeptide antigen expressed selectively by Purkinje cells reveals compartments in rat and fish cerebellum. J. Comp. Neurol. 291:53%552.

Chang, H.-T. (1952) Cortical neurons with particular reference to the apical dendrites. Cold Spring Harbor Symp. Quant. Biol. 17:189-202. Cochran, S.L., and J.T. Hackett (1977) The climbing fiber afferent system of the frog. Brain Res. 181:362-367. Colonnier, M., and C. Beaulieu (1985)An empirical assessment of stereological formulae applied to the counting of synaptic disks in the cerebral cortex. J. Comp. Neurol. 231:175-179. Crick, F. (1982)Do dendritic spines twitch? Trends in Neurosci. 5t44-46. Diamond, J., E.G. Gray, and G.M. Yasargil (1970) The function of the dendritic spine: An hypothesis. In P. Andersen and J.K.S. Jansen (eds): Excitatory Synaptic Mechanisms. Oslo: Universitet Forlaget, pp. 213222. Eccles, J.C., M. Ito, and J. Szentagothai (1967) The Cerebellum as a Neuronal Machine. Springer Verlag, Berlin-Heidelberg-New York, pp. 335. Fair&, A., A. Peters, and J. Saldanha (1977)A new procedure for examining Golgi impregnated neurons by light- and electronmicroscopy. J. Neurocytol. 6:311-337. Finger, T.E. (1978a) Cerebellar afferents in teleost catfish (Ictaluridae).

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