5′-Nucleotidase of cerebellar molecular layer: Reduction in purkinje cell-deficient mutant mice

Developmental Brain Research, 29 (1986) 93-100 Elsevier

93

BRD 50442

5'-Nucleotidase of Cerebellar Molecular Layer: Reduction in Purkinje Cell-Deficient Mutant Mice* DOUGLAS T. HESS 1and ARTHUR HESS2

1Department of Psychology E25-634, MassachusettsInstitute of Technology, Cambridge, MA 02139 and 2Departmentof Anatomy, Rutgers Medical School, Universityof Medicine and Dentistry of New Jersey, Piscataway, NJ08854 (U.S.A.) (Accepted March 25th, 1986)

Key words: cerebellum - - 5'-nucleotidase - - Bergmann glia - - enzyme induction

The molecular layer of the cerebellar cortex contains high levels of the enzyme 5'-nucleotidase (EC 3.1.3.5), localized predominantly to Bergmann glia. In the present study, histochemical methods have been employed to examine the distribution of cerebellar 5'-nucleotidase in mice of two separate mutant strains in,which Purkinje cells are eliminated selectively. In homozygous 'Purkinje cell degeneration' (pcd) and 'nervous' (nr) mice, molecular layer 5'-nucleotidase is greatly reduced and residual enzyme activity in colocalized with surviving Purkinje cells. These results suggest strongly that the expression of 5'-nucleotidase activity by Bergmann glia is under the inductive influence of their associated Purkinje cells.

INTRODUCTION In 1963, Scott 2°,21 showed with histochemical methods that the molecular layer of the cerebellar cortex in the mouse contains high levels of the enzyme 5'-nucleotidase, distributed in alternating longitudinal bands of relatively light and dark staining. Additional studies showed that high levels of 5'-nucleotidase occur in the cerebellar molecular layer in a number of species, with a banded distribution reported in rat and shrew in addition to the mouse 14,23. The banded distribution of 5'-nucleotidase constituted the first evidence for a compartmentalization of the cytoarchitectonically uniform cerebellar cortex beyond the gross division into vermis, pars intermedia and hemisphere. Subsequently, a large number of experimental anatomical studies have demonstrated that both the olivocerebellar and corticonuclear projections are organized into zones according to a modular architecture in which the basic unit is a longitudinally arrayed set of Purkinje cells sharing afferent

and efferent connections (see ref. 26 for a recent review). It was not possible, based upon light microscopic examination of histochemical material, to assign definitively a cellular localization to 5'-nucleotidase within the cerebellar molecular layer 2°-22. However, given the well-characterized cytoarchitecture of the cerebellar cortex 19 and the association of 5'-nucleotidase with cellular elements which extend through but are restricted to the molecular layer 2°,21, there exists a limited set of candidate cell types. The best of these candidates is the Bergmann glial or Golgi epithelial cell, the predominant glial cell type of the molecular layer 19. The cell bodies of these specialized astrocytes are distributed continuously within the plane of the Purkinje cell layer and their processes extend through the thickness of the molecular layer where extensive membrane elaborations place them in intimate association with the longitudinally oriented dendritic arbors of Purkinje cells ~9. Cytochemical and biochemical studies have in fact shown that 5'-nu-

* These results have been published partly in abstract form (ref. 6). Correspondence: D.T. Hess, Department of Psychology E25-634, Massachusetts Institute of Technology, Cambridge, MA 02139, U.S.A. 0165-3806/86/$03.50 O 1986 Elsevier Science Publishers B.V. (Biomedical Division)

94 cleotidase activity within the cerebellar cortex is predominantly astrocytic in origin (refs. 7, 10 and 11, but see ref. 14). Thus, the longitudinal compartmentalization of molecular layer 5'-nucleotidase appears to represent a differential expression of this enzyme by sets of Bergmann glia (and possibly by other glial cell types as well) according to the modular organization of the cerebellar cortex. This is the only example of which we are aware where the characteristics of glia within a population may vary regularly in accordance with the functional architecture of their resident central nervous system locus. It might be suggested that the differential expression of 5'-nucleotidase activity by groups of Bergmann glia is related causally to their association with functionally differentiated sets of Purkinje cells, a possibility in keeping with the intimate morphological relationship between the two cell types. To test the specific hypothesis of a role for Purkinje cells in regulating the expression of 5'-nucleotidase by Bergmann glia, we have employed histochemical methods to examine the distribution of 5'-nucleotidase activity in mutant mice in which Purkinje cells are eliminated selectively. We report here that in homozygous pcd and nr mice, molecular layer 5'-nucleotidase is greatly reduced and residual enzyme activity is colocalized with surviving Purkinje cells. These results suggest strongly that the expression of 5'-nucleotidase activity by Bergmann glia is under the inductive influence of their associated Purkinje cells. MATERIALS AND METHODS

Homozygous pcd/pcd (C57BL/6J) and nr/nr (C3HeB/FeJ) mice were obtained along with heterozygous littermates from Jackson Laboratories (Bar Harbor, Maine). Mutant mice were examined between 30-90 (pcd) or 50-94 (nr) days of age. Normal adult mice of the BALB/c strain were also employed. Transverse sections were cut in a cryostat at 20 ,urn thickness, dried onto glass slides and fixed by immersion for 2 min in 10% formalin-1% calcium chloride. Sections were reacted for 5'-nucleotidase following Naidoo 16 and Scott 22. The reaction medium contained 1 mM 5'-adenosine monophosphate (5'AMP) and 2 mM lead acetate in 50 mM sodium succinate buffer (pH 7.2). Some sections from normal mice were incubated with 5'-guanosine monophos-

phate (5'-GMP) in place of 5'-AMP as substrate. After incubation overnight at 4 °C (which yielded cleaner sections than shorter incubations at higher temperaturesL secuons were developed by exposure to dilute ammonium sulphide, immerslvd in 10% formalin-1 % calcium chloride for an addional t0 mins. dehydrated, cleared and coverslipped. Selected alternate sections were stained for Nissl substance. Control sections incubated without substrate showed little reaction product in the cerebellar molecular laver RESULTS

5'-Nucleotidase histochemistry in normal mice Reaction product indicative of 5'-nucleotidase actiwty is distributed discontinuously within the molecular layer of the mouse cerebellum, forming a series of alternating dark and light bands which run orthogonal to the long axes of the folia 2°'2l (Fig. 1A). Reflecting the association of the enzyme with the processes of Bergmann glia. the distinctly striated darkly staining bands extend from the Purkinje cell layer to the pial surface (Fig. 1A). Most molecular layerreaction product was localized diffusely on higher-power examination and could not be assigned to specific cellular elements. The Purkinje cell layer is delineated by staining of medium intensity in section reacted for 5'-nucteotidase with 5'-AMP as substrate (Fig. 1A. Bt. Reaction product is contained in many Purkinje ceils, and in numerous small cells which were identified as Bergmann glia based primarily upon their location within the Purkinje cell layer, or as granule cells immediately adjacent to the Purkinje cell layer (Fig. 1BI. In contrast to the banded distribution of reaction product within the molecular layer, staining within the Purkinje cell layer is uniform across the folia (Fig. lAI. Most granule cells are lightly stained, medium staining of neurons and neuropil delineates the deep cerebellar nuclei, and moderate activity is seen within the cerebellar white matter (Fig. 1A). In sections reacted for 5'-nucleotidase with 5 GMP as substrate, a pattern of molecular layer banding is seen which is identical to that observed after staining with 5'-AMP as substrate (Fig. IC), although reaction product is considerably less dense with 5'-GMP than with 5'-AMP after equal incuba-

95

Fig. 1. Histochemicallocalizationof 5'-nucleotidase activityin the cerebellum of the normal mouse. A: sectionillustrating distribution of reaction product after incubation with 5'-AMP as substrate. B: higher-power view illustrating distribution of reaction product in Purkinje cell layer, g, granule cell layer; m, molecular layer. C: distribution of reaction within the cerebellar cortex after incubation with 5'-GMP as substrate. Scale bars, 500~m in A and C, 50/~min B.

tion times. No activity is evident within the Purkinje cell layer after incubation with 5'-GMP as substrate (Fig. 1C). Staining outside the molecular layer is seen predominantly in association with blood vessels, and in the white matter where activity has a reticular appearance suggestive of an interfascicular distribution. 5'-Nucleotidase histochemistry in mutant mice pcd And nr are separate mutations, each of which leads to the selective loss of Purkinje cells in homozygous mutants 15,25. It has been reported that loss of Purkinje cells is nearly complete within the cerebellar hemisphere of nr/nr mice by about two months of

age with less complete elimination in the vermis, after which time little further change occurs 25. We found in nr/nr mice of 50-94 days of age that 5'-nucleotidase activity was greatly reduced and uniform within the molecular layer of the cerebellar hemisphere with no evidence of more darkly staining bands (Fig. 2A), in conjunction with the selective and virtually complete elimination of Purkinje cells (Fig. 2B). The distribution of reaction product in the granule cell layer, white matter and deep nuclei of nr/nr mice was indistinguishable from normal, and the distribution of reaction product in sections from heterozygous littermates processed in parallel with those from nr/nr mice did not differ noticeably from

96

Fig 2. Histochemical localization of 5'-nucleotidase activitv m the cerebellum of the nr:nr mouse. A: section illustr~ltingelimination oi molecular layer 5'-nucleotidase activity in the cerebellar hemisphere of a nr/nr mouse of'~4 days of age B: higher ;~owerwew of closely spaced Nissl-stained section illustrating selective elimmanon of Purkinje cells. Arrows indicate juncture of granule cell and molecular layers. C: residual molecular layer 5'-nucleotidase activit~ in a folium of the posterior vermis of a nr/nr mouse i~f94 days ol agt.. D: higher-power view of adjacent Nissl-stained section illustrating cluster of surviving Purkinje cells in the folium depicted in C Sc~dc bars. 250 um in A and C. 100 um in B. 50 um in D.

that seen in normal mice. The elimination of Purkinje cells and of molecular layer 5'-nucleotidase activity was not complete within the cerebellar vermis of nr/nr mice. Fig. 2C illustrates residual 5'-nucleotidase activity distributed in patches within the molecular layer of a foliurn of the posterior vermis of a nr/nr mouse of 94 days of age. Such vestigial bands were lightly stained in comparison with the bands of enzyme activity seen in the normal mouse (high contrast prints were used in Fig. 2A. C). A comparison with Nissl-stained sections revealed that residual molecular layer enzyme activity was present only in folia where surviving Purkinje cells, distributed in clusters, could be identified. Fig.

2D shows a cluster of Purkinje cells underlying the rightmost patch of molecular laver enzyme actwitv depicted in Fig. 2C. There was not a simple, one-toone correspondence between surviving Purkinje cells and enzyme activity. In particular, zdl Purkinje cells did not underly residual molecular laver reaction product: it should be noted that a substantial proportion of Purkinje cells are located under 5'-nucleotidase-poor regions of molecular laver in the normal mouse. It has been reported that the elimination of Purkinje cells is nearly complete throughout the cerebellum in p c d / p c d mice by about 3{) days of age 15. Cerebeilar sections from p c d / p c d mice of 4 5 - 9 0 days of

97

Fig. 3. Histochemical localization of 5'-nucleotidase activity in the cerebellum of the pcd/pcdmouse. A: section illustrating elimination of molecular layer 5'-nucleotidase activity in a pcd/pcdmouse of 90 days of age. This section through the cerebellar anterior lobe also passed through the posterior aspect of the inferior colliculi, which are stained darkly. B: higher-power view of closely spaced Nisslstained section illustrating selective elimination of Purkinje cells. Arrows indicate juncture of granule cell and molecular layers. C: 5'-nucleotidase activity in two adjacent folia of the posterior vermis of a pcd/pcdmouse of 30 days of age. Dorsal is up. See text for further details, g, granule cell layer: m, molecular layer. D: higher-power view of adjacent Nissl-stained section illustrating Purkinje cells present in the ventral folium depicted in C. Scale bars, 500~m in A, 100/~min B and C, 50/tm in D.

age reacted for 5'-nucleotidase revealed light and uniform staining within the molecular layer throughout the cerebellum, with no evidence of more darklystaining bands (Fig. 3A). Nissl-stained sections showed a selective and virtually complete loss of Purkinje cells (Fig. 3B). Staining for 5'-nucleotidase within the granule cell layer, white matter and deep nuclei ofpcd/pcd mice at all ages examined was indistinguishable from normal, and the distribution of 5'nucleotidase in cerebellar sections from heterozygous littermates did not differ significantly from that seen in normal mice. In some pcd/pcd mice of 30 days of age, the elimination of Purkinje cells and of molecular layer 5'-nucleotidase was not yet complete within the vermis.

Fig. 3C illustrates the distribution of 5'-nucleotidase activity in two adjacent folia of the posterior vermis of a pcd/pcd mouse of 30 days of age. Within the field of view shown, the molecular layer still stains relatively darkly in the ventral folium and in a short segment of the dorsal folium. Darkly stained Purkinje cells are evident throughout the ventral folium, but are virtually absent from the dorsal folium (Fig. 3C). An adjacent Nissl-stained section provided confirmation that Purkinje cells were still present in the ventral folium, although at reduced cell density (Fig. 3D). Purkinje cells in the dorsal folium had degenerated fully, but molecular layer 5'-nucleotidase activity had not yet been eliminated.

98 DISCUSSION The main findings reported here are that 5'-nucleotidase activity of the cerebellar molecular layer is greatly reduced in mutant mice which lack Purkinje cells selectively and that residual activity is colocalized with surviving Purkinje cells. These findings suggest strongly that the expression of 5'-nucleotidase activity by Bergmann glia (and possibly by other glial cell types as well) is under the inductive influence of their associated Purkinje cells. Our results do not bear directly on the question of whether this influence is exerted at the level of transcription or translation, or posttranslationally. One alternative explanation for these findings would be that both the nr and pcd mutations are associated with some general deficit in the expression of 5'-nucleotidase. This is clearly not the case. as the localization in pcdlpcd and nr/nr mice of 5'-nucleotidase elsewhere in the brain, and in the cerebellum except as illustrated, is indistinguishable from normal. A second alternative explanation would be that Bergmann glia as well as Purkinje cells are depleted in the cerebellum of nrmr and pcd/pcd mice We arc aware of no reports of a depletion of Bergmann glia in the nr or pcd cerebellum and the existence of an apparently normal complement of Bergmann gtia in the cerebellum of both nr/nr andpcdlpcd mice has recently been shown directly with immunohistochemical staining for glial fibrillary acidic protein 2. A third alternative explanation for our results would be that the deficit in expression of 5'-nucleotidase is a direct effect of the nr and pcd mutations on the Bergmann glia. Although this possibility cannot be rejected based on the present results, it does not seem likely that two separate and distinct mutations which lead to the death of Purkinje cells would have identical direct or intrinsic pleiotropic effects on the Bergmann gila.

Comments on the histochemical localization of cerebellar 5'-nucleotidase In sections of mouse cerebellum reacted for 5'-nucleotidase with 5 ' - A M P as substrate, many cells within the Purkinje cell layer are stained. However. activity within the Purkinje cell layer is uniform across the folia, in contrast to the banded distribution of reaction product within the molecular layer. In addition.

substitution of 5 ' - G M P for 5 ' - A M P as substrate ehm-, inates staining in the Purkinje cell laver but yields an identical pattern of banding within the molecular la~er. Thus. there appears to be at least in part a dissociation between 5'-nucleotidase acfiwty within the molecular layer and activity seen m the Purkinje cell layer after incubation for 5'-nucleotidase with
Comparison with relevant previous findings A number of examples have been described in which the interaction of gila with neurons plays an obligatory role in the expression of gtial properties 24 In particular, studies in the quail have shown that the production by dorsal root ganglion ghal precursors of the protein S-100 is dependent upon their interaction with ganglionic sensorv neurons ~" Similarly. the cortisol-mediated induction of glutamine svnthetase production by Mfiller cell gliocytes of the chick retina requires thmr interaction with retinal neurons ~2 These interactions are characterized by the importance of direct contact between neurons and glia s~= A similar role for cell surface interactions might be suggested for the regulation of expless~on of 5'-nucleotidase activity by Bergmann gim, given the intimate morphological association between the Bergmann glia and Purkinjc cells and the colocalization of molecular laver 5'-nucleotidase and surviving Purkinje cells in Purkinje cell-deficient mutant mice, in work done contemporaneously with thai reported here, Fisher 2 nas shown that the expression by Bergmann gila of the enzyme sn-glycerol-3-phosphate dehydrogenase ( G P D H ) is under the inductive influence of Purkinje cells. In thai study, immunohistochemical staining for G P D H in Purkinje celt-deficient mutant mice yielded results similar to those re-

99 ported here for 5'-nucleotidase, although the time course of disappearence of G P D H immunoreactivity subsequent to the elimination of Purkinje cells is apparently more prolonged 2. The present results and those of Fisher 2 demonstrate that the functional state of the Bergmann glia is determined significantly through interaction with their associated Purkinje cells.

Functional implications of Purkinje cell-Bergmann glia interaction 5'-Nucleotidase may play a number of roles as an important enzyme in nucleotide metabolism 13,17. In neural as in other tissue, 5'-nucleotidase is present predominantly in membrane-bound form and is considered to function in large part as an ectoenzyme, converting nucleotides to the corresponding nucleosides within the extracellular milieu 11,13A7.This mode of function of 5'-nucleotidase may be of particular importance with respect to neuronal purinergic receptors 1. It has been shown recently that the molecular layer of the cerebellar cortex is well-endowed with specific adenosine receptors of the Al-type 3'4. These receptors are not present in the agranular cerebellum of homozygous 'weaver' (wv) mice 3,27, which suggests that they are presynaptic receptors associated with the parallel fiber-Purkinje cell synapse. This suggestion is supported by the finding that adenosine suppress the excitatory influence of parallel fibers on Purkinje cells via a presynaptic mechanism of action 9. A correlation between high levels of 5'-nucleotidase and specific adenosine receptors does not hold in all brain areas. In particular, the superficial layers of the superior colliculus are well endowed with A r t y p e adenosine receptors 4, which appear to be presynaptic in location as they are eliminated by eye removal 3, yet we have observed that 5'nucleotidase levels are conspicuously low in the superficial superior colliculus (unpublished observation). In addition, it should be noted that the distribution of Al-type adenosine receptors is uniform across the cerebellar folia in mouse and rat 3,4, in contrast to the banded distribution of 5'-nucleotidase. Nonetheless, 5'-nucleotidase is frequently found in perisynaptic locations in the molecular layer7,1°,11 and to the extent that 5'-nucleotidase influences adenosine levels at adenosine receptors associated with the parallel fi-

ber-Purkinje cell synapse, the present results illustrate a complex interaction wherein the postsynaptic neuron exerts inductive control over a (glial) enzymatic mechanism which serves to regulate levels of a presynaptic modulator. We have previously reported 6 that in the agranular wv/wv cerebellum, 5'-nucleotidase remains at significant levels in the molecular layer in the absence of parallel fibers, i.e. in the absence of adenosine receptor-bearing elements 3,27. The findings reported here have some bearing on the question of the origin of the banded distribution of 5'-nucleotidase in the cerebellar molecular layer of the mouse and rat 21,23. Studies employing immunohistochemical staining for the peptide motilin TM and for an unidentified antigen with antibodies to cerebellar tissue produced by monoclonal methods 5, have provided evidence that Purkinje cells in rat and mouse may be grouped into longitudinal zones based on intrinsic differences between Purkinje cells. The present results suggest that Purkinje cells may also differ systematically in their inductive influence on Bergmann glia, according to the longitudinal zonal organization of the cerebellum. The basis of this difference is unknown, although cell surface characteristics are presumably involved. Similarly, it is not clear why there exist large species differences in the topographic distribution of molecular layer 5'-nucleotidase; the banding pattern in rat differs from that in mouse and in the cat 5'-nucleotidase activity is distributed uniformly within the molecular layer 14,23. It is possible that developmental studies will prove to be of use in determining the characteristic(s) of Purkinje cells through which is exerted their inductive influence on Bergmann glia. We have examined the development of 5'-nucleotidase activity in the normal mouse cerebellum from birth through adulthood (unpublished observations). In the neonatal cerebellar cortex, 5'-nucleotidase activity is restricted to and evenly distributed within the developing molecular layer. No evidence of a banded distribution may be discerned until about postnatal day 14, after which time conjoint increases and decreases in staining intensity gradually yield the adult pattern of distribution. These observations suggest that the inductive influence of Purkinje cells on the expression of glial 5'-nucleotidase activity is exerted according to a specific developmental timetable.

100 ACKNOWLEDGEMENTS

G M 0 7 4 8 4 a n d M H 1 5 7 6 1 . T h e a u t h o r s t h a n k P. A d a m o a n d A . B r e t s c h n e i d e r for t h e i r i n v a l u a b l e assis-

S u p p o r t e d in p a r t by N I H G r a n t s H L 3 1 3 6 7 a n d

tance.

NS21469, w i t h a d d i t i o n a l s u p p o r t f r o m E Y 0 0 1 2 6 ,

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