THE JOURNAL OF COMPARATIVE NEUROLOGY 315:85-97 (1992)

Development of Glutamic Acid Decarboxylase-Immunoreactive Elements in the Cerebellar Cortex of Normal and Lurcher Mutant Mice JOHN A. HECKROTH Indiana University School of Medicine, Terre Haute Center for Medical Education at Indiana State University, Terre Haute, Indiana 47809

ABSTRACT The development of glutamic acid decarboxylase-immunoreactivity (GAD-IR) in cells, fibers, and varicosities of the cerebellar cortex has been examined by light microscopy in normal and lurcher mutant mice between postnatal day 3 and 30 (P3-P30). Purkinje cell morphology was demonstrated in adjacent sections by using an antiserum to the 28Kd vitamin D-dependent calcium binding protein (CaBP). In early postnatal lurcher mice, but not in normal littermates, GAD-IR fibers, presumably Purkinje cell pseudopodia, invade the external granular layer. The plexus of CaBP-IR axons in the internal granular layer is much less complex in lurcher mice than in normal littermates, even before the onset of lurcher Purkinje cell degeneration at P8. In normal mice, GAD-IR fibers encapsulate Purkinje cell somata by P15. Lurcher Purkinje cells, in contrast, receive scattered contacts by GAD-IR puncta and possess a “cap” of such elements surrounding the primary dendrite and apical soma. Pinceau formations, visible as a knot of GAD-IR puncta hanging from the base of Purkinje cells in normal P15 mice, are not present in lurcher littermates. “Empty baskets” or collapsed pinceau formations in regions devoid of Purkinje cells are not revealed by anti-GAD immunohistochemistry in the P17-P30 lurcher cerebellar cortex. Key words: cerebellum, neurological mutant, Purkinje cell

The lurcher mouse, originally isolated and described by Philips (’601, displays a characteristic locomotor ataxia (Fortier et al., ’87) as the result of a semidominant mutation (Lc) in linkage group XI on chromosome 6. Initial neuropathological examinations (Caddy and Biscoe, ’75; Wilson, ’75; Wilson, ’76; Swisher and Wilson, ’77) revealed a striking cerebellar atrophy based on the loss of Purkinje and granule cells, and a severe inferior olivary atrophy. Quantitative studies (Caddy and Biscoe, ’79) revealed the absence of virtually all Purkinje cells, 90% of the cerebellar granule cells, and 60-75% of inferior olivary neurons in adult lurcher mice. Studies of Lcl+-+ / + chimeras (Wetts and Herrup, ’82a,b) established that the loss of lurcher granule and olivary neurons was the result of retrograde transneuronal degeneration induced by the elimination of Purkinje cells by the lurcher gene. Despite this demonstration of the restricted nature of the direct effect of the lurcher gene, which permits relatively clear interpretation of pathological changes, few studies have described further details of the lurcher phenotype. The recent report by Norman et al. (’91) indicates that knowledge of the gene product affected by Lc may be o 1992 WILEY-LISS, INC.

forthcoming. A comprehensive knowledge of the phenotypic effects of this mutation will be necessary in order to accurately describe the full range of influence of Lc, and hence to correctly interpret the role of its normal allele in brain development. The recent description of an important defect in olivocerebellar fiber development in lurcher (Heckroth et al., ’90) suggests that some significant aspects of the lurcher phenotype may remain unrecognized. It is the aim of this report to identify previously undescribed developmental expressions of the lurcher phenotype in order to facilitate the eventual application of molecular genetic results to the interpretation of pathological development in this mutant. Previous ultrastructural observations (Heckroth et al., ’90) led to the suggestion that, in addition to defective climbing fiber development, defects in basket cell axon maturation might also exist in lurcher. The present study Accepted September 23,1991. Address reprint requests to John A. Heckroth, Indiana Univ. School of Medicine, Terre Haute Ctr. for Medical Education at Indiana State Univ., 135 Holmstedt Hall, Terre Haute, IN 47809.

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antiserum was used at a dilution of 1:100,000 in PBS with 0.3% Triton X-100 (PBSiTriton) containing 10% normal goat serum (NGS). Controls for this antiserum consisted of the substitution of normal rabbit serum for the specific antiserum, and the omission of secondary antibody or avidin-peroxidase in subsequent steps. Each control condition abolished all staining, other than non-specific staining of erythrocytes. Sheep anti-GAD was provided through the Laboratory of Clinical Science, NIMH, where it was developed under the supervision of Dr. Irwin J. Kopin with Drs. Wolfgang Oertel, Donald E. Schmechel, and Marcel Tappaz (Oertel et al., '82; Wu et al., '82). This antiserum was used at a dilution of 1:4000 in PBS, or PBSitriton, containing 10% normal rabbit serum (NRS). Controls for this antiserum consisted of the substitution of preimmune serum for the specific antiserum, or of the omission of secondary antibody or avidin-peroxidase in subsequent steps. Each control condition abolished all staining, other than non-specific staining of erythrocytes. Sections were processed free-floating in glass shell vials. Following three 10 minute rinses in PBS (or PBS/triton) and 30 minutes in 10% blocking serum, sections were incubated for 15-20 hours in primary serum. A one hour incubation in biotinated secondary antibody (Vector) diluted 1 : l O O in 10% blocking serum was preceded and followed by PBS rinses. Sections were treated for one hour MATERIALS AND METHODS in ABC reagent (Vector),followed by three 10 minute rinses Animals in 0.1 M tris buffered saline (TBS, pH = 7.4), and then Normal and lurcher littermates (BGCBA) were obtained developed in 0.05% diaminobenzidine/0.003% hydrogen from a colony maintained in the Terre Haute Center for peroxide in TBS (10-20 minutes). After thorough rinsing Medical Education animal facility, bred from stock origi- with TBS, the sections were mounted on gelatin-coated nally purchased from Jackson Labs. The Miwhallele, linked microscope slides and allowed to dry in air. Coverslips were with Lc, has served as a marker for mutant animals before then applied with Cytoseal60. The limitations of the immunohistochemical method are, the onset of the characteristic ataxia. Male MiwhLc/+ + animals were bred with + + / + + females. Carriers of the by now, well-known. Although the GAD and CaBP antisera MiWhLcchromosome are easily identified at birth by the utilized in this study have been well-characterized, and lighter pigmentation associated with the Miwhallele. This staining patterns in normal adult cerebellum throughly system provides a 90% assurance of the Lcl+ genotype described, some question may arise regarding antigen prior to the onset of overt phenotypic manifestations localization in developing tissue, and in mutant mice. The (Phillips, '60). The presence of the Miwhgene has no known possible presence in these tissues of cross reactive antigens not present in normal adult cerebellum must be acknowleffect on the Lcl+ phenotype (Wilson, '76). Mutant and normal littermate pairs were sacrificed at edged. Results are referred to as GAD or CaBP immunorepostnatal day 3 (P3), P5, P6, P8, P9, P11, P13, P14, P15, actiuity, with the implied understanding that the nature of the immunoreactive antigens in developing and mutant P17, P21, and P30 (13 pairs). cerebella has not been verified by biochemical means. It must also be remembered that the developmental appearTissue preparation Animals were sacrificed with Avertin and perfused with ance of immunoreactivity signifies only the accumulation of normal saline containing 0.1% procaine, followed by 4% sufficient quantities of antigen for detection by this method. paraformaldehyde in 0.12 M PO, buffer (pH = 7.4). Follow- Conversely, the absence of immunoreactivity indicates only ing in situ postfixation for 15-20 hours at 4"C, the brains insufficient quantities of antigen for detection, and not were dissected from the skull while immersed in phosphate necessarily the absence of morphological entities. buffered saline (PBS; pH = 7.4) and blocked for sagittal sectioning. Sections were cut at a thickness of 50 Fm on a RESULTS Lancer vibratome and collected in PBS where they were stored prior to immunohistochemical processing. The anti-GAD immunohistochemical staining protocol used in this study reveals both terminals and cell bodies Immunohistochemistry when Triton-X is omitted from the incubation media (Fig. Adjacent sections were processed for immunohistochem- 1A). The addition of Triton-X (Fig. 1B) provides increased istry with one of two primary antibodies. Rabbit anti- visibility of IR fibers and terminals, presumably by invitamin D dependent calcium binding protein (CaBP) was creased tissue penetration by reagents, but abolishes cell generously provided by Dr. Anthony Norman (University of body staining in all but the most intensely labeled cells. At California, Riverside). Preparation and characterization of each age examined adjacent sections were prepared and this antiserum has been reported in detail previously (Roth compared by these two complementary protocols. The et al., '81; Garcia-Segura et al., '84; Fournet et al., '86). The GAD-IR staining pattern observed in normal mature cere-

was initiated in order to examine the basket cell axons in lurcher, by using an antiserum to glutamic acid decarboxylase (GAD) as a marker. In normal animals, the strong GAD-immunoreactivity of basket cell axons permits clear visualization of the characteristic pericellular baskets and pinceau formations surrounding Purkinje cell somata and axons (Oertel et al., '81). In addition, the axons and cell bodies of the Golgi and stellate cells, and of the Purkinje cells themselves, are GAD-immunoreactive (IR). Previous studies have shown that GAD-IR elements persist in the adult lurcher cerebellar cortex (Biscoe et al., '84), and the distribution of GABA receptors in this mutant has been described (Fry et al., '85). Although the developmental appearance of GAD in the cerebellum has been examined biochemically (Coyle and Enna, '76; Hadjian and Stewart, '77; Unsworth et al., '801, no comprehensive study of the development of GAD-like-IRis available. This report describes the appearance of GAD-immunoreactivity in normal and lurcher mice between P3 and P30, and correlates these results with Purkinje cell morphology as revealed by an antiserum to the 28Kd vitamin D-dependent calcium binding protein (CaBP). Abnormalities in the formation of GAD-IR pericellular baskets and pinceau formations in lurcher are demonstrated, and other early phenotypic expressions of the lurcher gene are revealed.

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Fig. 1. Photomicrographs of P21 normal mouse cerebellar cortex. A: Glutamic acid decarboxylase-immunoreactivity (GAD-IR) without Triton X-100, demonstrating cell body staining in Golgi (G), stellate (s), and basket cells, and the characteristic pattern of terminal staining observed in mature cerebellar cortex. B: GAD-IR in the presence of Triton X-100, showing the loss of cell body staining, and the increased

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density of immunoreactive terminals compared with "A". C: Calcium binding, protein immunoreactivity (CaBP-IR) in an adjacent section to A and B illustrating the fine details of Purkinje cell cytology revealed by this marker, including the axons and varicosities of the infraganglionic plexus. Scale bar in A also refers to B and C. g, granular layer; m, molecular layer; pc, Purkinje cell layer.

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bellar cortex in this study (Fig. 1A and B) reproduces in every detail that described previously in other rodent species (Saito et al., '74; Oertel et al., '81). Immunochemical staining with anti-CaBP in both normal and lurcher mice reveals Purkinje cell morphology with great clarity and detail (Fig. lC), as previously reported (Roth et al., '81; Garcia-Segura et al., '84; Fournet et al., '86). Although these reports also describe staining of cerebellar Golgi cells, it was not observed in the present study, perhaps because of the high dilution of antiserum employed (1:100,000). At all ages studied, Purkinje cell somata, dendrites, and axons are intensely immunoreactive. Purkinje cell morphology undergoes a well-described transformation over the first two postnatal weeks in mice (Meller and Glees, '69; Hendelman and Aggerwal, ,801, and these alterations in form are corroborated by the results of the present study. Also observed in this study are CaBP-IR fibers and varicosities constituting the infraganglionic plexus of the cerebellar cortex. These presumptive Purkinje cell axon recurrent collaterals are present at the earliest age examined (P3) in both lurcher and normal mice. CaBP-IR profiles, presumably Purkinje cell axons and terminals, are present in the cerebellar nuclei of normal mice at all ages examined, with no significant alterations in appearance over the developmental period under study. In lurcher, however, these immunoreactive elements become varicose at early ages, and disappear almost entirely by P30.

P3-P6 Normal. At P3 anti-GAD cell staining in the cerebellar cortex is weak, with only occasional Purkinje cells being visible. In the nascent internal granular layer (IGL), many GAD-IR fibers are present, forming an irregular network, with dense knots of immunoreactivity at points of intersection. Examination of adjacent sections processed for CaBPimmunoreactivity reveals the presence, already at P3, of a rich plexus of Purkinje cell axons, both in the IGL, and formingthe infraganglionicplexus (Fig. 2A). GAD-IR puncta and fibers are present among the Purkinje cells at this early age. These small puncta, more readily visible at P5 (Figs. 4A,C), are usually associated with the apical cytoplasm of the Purkinje cells and their pseudopodial extensions which radiate towards the external granular layer (EGL).Whether CaBP-IR puncta invade the Purkinje cell layer and nascent molecular layer cannot be determined because of the intense CaBP immunoreactivity of the Purkinje cell somata. By P5-P6, GAD-IR Purkinje cells are more consistently visible, but stain with variable intensity. GAD-IR Golgi cells are first observed in the IGL at P5. Lurcher. At P3, striking differences between the lurcher and normal cerebellum are already apparent. The CaBP-IR fiber plexus in the IGL is much less complex in lurcher (Fig. 2B) than in normal animals (Fig. 2A). In addition, GAD-IR fibers are frequently observed within the lurcher EGL during this period (Figs. 2D, 3A), but are rarely observed in normal mice (Fig. 2 0 . CaBP-IR fibers are also present in the lurcher EGL (Fig. 3B), thus implicating the Purkinje cells as a potential source of the GAD-IR fibers. The GAD-IR puncta observed in close association with Purkinje cell pseudopodia in the normal animals are also present in lurcher (Figs. 4B,D). Beginning on P5, the CaBP-IR fibers of the infraganglionicplexus, and those crossing the granular layer to enter the white matter, become varicose in

lurcher (Fig. 5A), although no abnormalities are observed in the lurcher cerebellar nuclei at this age (Fig. 5C).

P8-P9 Normal. Small, lightly GAD-IR cells located within the Purkinje cell layer and in the lower one-third of the molecular layer, presumably basket cells, are first observed at P8 (Fig. 6A). At this same age GAD-IR puncta become numerous throughout the molecular layer. The upper one-quarter of the molecular layer, however, remains relatively free of these GAD-immunopositivepuncta. Lurcher. Small GAD-IR cells also appear in the lurcher molecular layer at P8, but they are more frequent and more deeply staining than in normal animals, and are observed throughout the thickness of the molecular layer (Fig. 6B). The pathological alterations of the CaBP-IR axons initially observed in the cerebellar cortex at P5 (Figs. 5A,C), are found throughout the length of the axons, including their telodendria in the cerebellar and vestibular nuclei at P8 (Figs. 5B,D). The lurcher EGL is only about one-half as thick as in normal animals of the same age, and this layer still contains GAD-IR fibers, as observed in the younger specimens.

Pll-P15 Normal. Two major transformations in the pattern of GAD-immunoreactivityoccur during this period. First, in the granular layer, the disorganized network of fiber bundles and tangles which has persisted since the formation of the IGL is gradually replaced by the familiar pattern of rings of small IR puncta (Fig. 7E) characteristic of the mature condition (Saito et al., '74; Oertel et al., '82). As these rings increase in number, the immunoreactive fibers disappear. Second, the scattered GAD-immunopositive puncta apposed to the Purkinje cell somata become more numerous, and a plexus of large horizontal GAD-IR fibers above the Purkinje cell somata in the molecular layer becomes conspicuous. By P15 (Fig. 7A) the Purkinje cell somata are completely encapsulated by intensely GAD-IR profiles (Figs. 7C,E). The pinceau formation becomes visible during this period as a knot of GAD-IR profiles hanging from the base of many Purkinje cells (Figs. 7C,E). Lurcher. The maturation of GAD-IR elements in the granular layer proceeds normally in lurcher during this period (Fig. 7B). However, although GAD-immunopositive puncta apposed to the Purkinje cell somata are present in lurcher (see Figs. 4B,D), the complete encapsulation of Purkinje cell somata described in normal mice is not observed in lurcher. Instead, a discontinuous shell of unusually large (3-4 p,m) GAD-IR puncta is present, and an accumulation of these elements is usually found near the site of emergence of the primary dendrite (Figs. 7B,D). The main dendritic trunks of the lurcher Purkinje cells appear covered with these large GAD-immunopositive puncta as well. Well-developedpinceau formations, frequent and conspicuous in normal P15 mice (Fig. 7E), are never observed in lurcher (Fig. 7F).

P 17-P30 Normal. The qualitative pattern of GAD-immunoreactivity in normal mice changes very little during this period. The basket and pinceau formations become gradually more conspicuous, to become the dominant GAD-immunopositive elements in the mature cortex, while GAD-immunore-

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Fig. 2. Cerebellar cortex of P3 normal (A and C) and lurcher (B and D) mice, processed for CaBP-IR (A and B) or GAD-IR (C and D).Note the decreased complexity of the plexus of CaBP-IR axons in the lurcher granular layer (g) at this young age (B). Arrowheads in D indicate

GAD-IR processes within the external granular layer (egl) in lurcher, which are absent in normal mice (C). Scale bar in A also refers to B-D. pc, Purkinje cell layer.

activity of the Purkinje cell somata and dendrites becomes less intense. Lurcher. As lurcher Purkinje cells degenerate in large numbers during this period, considerable alterations in

cortical architecture and in the pattern of GAD-immunoreactivity occur (Fig. 8). In the granular layer, the GAD-IR puncta increase in size, and the rings of these elements appear to collapse inward. In regions devoid of Purkinje

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Fig. 3. Photomicrographs illustrating GAD-IR (A) and CaBP-IR (B)processes in the external granular layer of P3 lurcher cerebellar cortex. Scale bar in A refers also to B.

cells, the Purkinje cell layer remains visible as a pale staining lamina, with few GAD-IR elements. No “empty baskets” or persistent pinceau formations are observed.

DISCUSSION The present study has described two early phenotypic manifestations of the lurcher gene. First, abnormalities in CaBP-IR Purkinje cell axons may be observed, even in the youngest animals examined. At P3, a decreased complexity of the infraganglionic plexus is apparent in lurchers. By P8 this defect is quite pronounced, although elimination of Purkinje cells has not yet begun (Caddy and Biscoe, ’79). Once Purkinje cell degeneration begins (P8-PlO) the paucity of CaBP-IR fibers in the infraganglionic plexus becomes severe. Those CaBP-IR axons that do contribute to the infraganglionic plexus in lurcher show pathological changes in the form of large varicosities beginning on P5. Interestingly, the plexus of recurrent collaterals and proximal portions of Purkinje cell axons in the granular layer show these changes before they appear in the cerebellar nuclei. By P8, Purkinje cell axons exhibit varicosities throughout their length, including their ramifications in the cerebellar and vestibular nuclei. Early involvement of the axons has also been recently described in the hyperspiny mutant (Sotelo, ’90)although in this mutant patholog-

ical changes occur first in the terminal fields in the cerebellar nuclei, subsequently spreading toward the Purkinje cell bodies. A second early phenotypic expression of the lurcher mutation is the presence of GAD-IR profiles within the EGL at P3-6. The morphological similarity of the GADand CaBP-IR elements suggests that they represent the invasion of the IGL by Purkinje cell pseudopodial or axonal processes. Such processes are rarely observed in normal mice, but never penetrate as far into the EGL as is observed in lurcher. An interesting conjecture is that this morphological abnormality may underlie alterations in granule cell development observed in lurcher. Quantitative estimates (Caddy and Biscoe, ’79) indicate a loss of granule cellsprior to Purkinje cell degeneration (25%reduction at P4). This early reduction in granule cell number in the EGL could be produced by reduced proliferation of precursors in the EGL, or by the elimination of premigratory granule cells. The present report documents a reduction in EGL thickness (Fig. 61, confirming the observations of Swisher and Wilson (’771, who also provide autoradiographic evidence of proliferative anomalies in the lurcher EGL. In addition, these authors noted the presence of pyknotic debris in the molecular layer and EGL, further corroborating an early granule cell loss. Vogel and Herrup (’89) present “Hthymidine pulse labeling data which could be interpreted to

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Fig. 4. Cerebellar cortex of P5 normal (Aand C)and lurcher mice (Band D) illustrating GAD-IR elements. In A and B, GAD-IR Golgi cells, as well as a plexus of IR fibers and puncta, are present in the internal granular layer (g). In the Purkinje cell layer (pc), note the

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variable intensity of Purkinje cell staining. In C and D (enlargements of areas in A and B, respectively), note GAD-IR puncta in close apposition to Purkinje cell filopodia and somata. Scale bars in A and C also refer to Band D, respectively. egl, external granular layer; P, Purkinje cell.

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Fig. 5. Cerebellar cortex (A and B) and nuclei (C and D) in P5 (A and C) and P8 (B and D)lurcher mice processed for CaBP-IR. Note axonal varicosities on proximal portions of Purkinje cell axons (arrowheads in A), but not in the cerebellar nuclei (C) at P5. By P8 these

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pathological swellings are present throughout the length of Purkinje cell axons (B), including portions within the cerebellar (D) and vestibular nuclei. g, granular layer; egl, external granular layer.

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Fig. 6. GAD-IR elements in P8 cerebellar cortex of normal (A) and lurcher (B)mice. Note the presence of small immunoreactive cells (arrows) in the lower molecular layer in A, but throughout the thickness of the molecular layer in B. The external granular layer (egl) is thicker in normal than lurcher mice at this age. P, Purkinje cells.

indicate an early cessation of granule cell proliferation in lurcher, and perhaps a shift in proliferative kinetics of the granule cell precursors (an earlier peak in granule cell production?), although short survival pulse labeling studies are required in order to interpret these data accurately. Such precocious EGL abnormalities appear difficult to reconcile with the demonstration (Wetts and Herrup, '82a) that Lc directly affects only the Purkinje cells, as an early granule cell loss cannot be the result of retrograde transneuronal degeneration. Intrinsic Purkinje cell abnormalities could, however, contribute to abnormalities in granule cell development through trophic interactions with cells of the EGL. The potential for such an interaction has been suggested by other investigators (Mallet et al., '76; Das and Pfaffenroth, '77; Mariani et al., '77; Landis and Sidman, '78; Herrup and Mullen, '791, and the regulation of granule cell number by Purkinje cells through an unknown mechanism has been demonstrated (Wetts and Herrup, '83; Herrup and Sunter, '87; Chen and Hillman, '89). It is thus conceivablethat the GAD-IR processes observed in the EGL of lurcher may somehow inhibit granule cell proliferation, or reduce viability of premigratory granule cells. If so, the

lurcher mutant mouse may prove to be a valuable model system in which to analyze the nature of this important type of interaction. The data presented in this report cannot determine whether EGL abnormalities permit or facilitate the abnormal invasion by GAD-IR fibers, or as is suggested above, the invading fibers induce EGL abnormalities. Currently available data (Wetts and Herrup, '82a), however, incriminate the lurcher Purkinje cell as the only site of direct gene action in this mutant cerebellum. In addition to these early effects of Lc, the results of this study provide good evidence that the conspicuous pericellular baskets and pinceau formations (the terminals of the basket cells), associated with the Purkinje cells in normal animals, undergo abnormal development in this mutant, as previously suggested (Heckroth et al., '90). This contention is supported by three findings: (I) lurcher Purkinje cell somata and initial segments are not completely enveloped by GAD-IR terminals at any postnatal age between P3 and P30; (2)persistent GAD-IR baskets and pinceau formations are not observed followingthe elimination of Purkinje cells, as is observed in other mutants which suffer large scale

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~

Figure 7

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including the Purkinje cells themselves, the stellate cells, Golgi cells, or extracortical sources. The virtual absence in lurcher of Purkinje cell recurrent collaterals at Pll-15 (as revealed by CaBP immunochemistry),makes Purkinje cells themselves an unlikely origin for these immunoreactive varicosities. Extracortical sources of GAD-IR mossy fibers have been suggested in normal animals (Batini et al., ’89), but seem an unlikely source for the rich system of Purkinje cell-associated GAD-IR puncta observed in the developing lurcher cerebellum. The Golgi cells in lurcher appear to enter into normal relationships within the cerebellar glomeruli, and GAD-IR puncta persist in these structures in adult lurcher mice. The simultaneous contribution of Golgi cells to this normal system, and to an abnormal relationship with Purkinje cell somata, seems a remote possibility. Finally, the potential contribution of the stellate cells to the GAD-IR caps on Purkinje cells in lurcher must be considered. Unfortunately, the initial appearance of GAD-IR microneurons throughout the thickness of the lurcher molecular layer, rather than only in its lower third, blurs the distinction between the basket and stellate cell populations. If proliferative anomalies do occur within the lurcher EGL, then another important distinction between these neuron classes (timing of final mitosis) may be obscured in this mutant. Clearly, the discrimination of the lurcher basket and stellate cell populations is problematic. The GAD-IR caps and their cells of origin in lurcher are presumed to be homologous with the pericellular baskets and basket cells in normal mice, on the basis of general similarities in form and development, although this tentative conclusion awaits further confirmation. Ultrastructural studies in progress may clarify this issue. The temporal coincidence of basket formation by the Fig. 8. GAD-IR elements in P21 lurcher cerebellar cortex. Note the basket cell axons and of olivocerebellar fiber ascent in numerous GAD-IR puncta in the molecular layer. GAD-IR stellate(s) normal animals, together with defects in both these Purand possibly basket (b?) cells are present as well. Golgi cells (G) are kinje cell afferent fiber types in lurcher, suggests an imporvisible in the granular layer, as are their presumed terminals in tant causal relationship between these developmental cerebellar glomeruli (rings of GAD-IR puncta). Note the conspicuous events. Manipulations of one or the other afferent type in immunonegativity of the former Purkinje cell layer (pc), and the normal animals, however, reveal the potential for indepenabsence of persistent “empty baskets” or pinceau formations. dent development of these two systems. Experiments involving the elimination of olivocerebellar fibers by neonatal Purkinje cell loss (Sotelo and Triller, ’79; Wassef et al., ’86); pedunculotomy (Sotelo and Arsenio-Nunes, ’76) demonand (3) GAD-IR elements form abnormal “cap” structures strate that, in the absence of climbing fibers, Purkinje cell on lurcher Purkinje cell somata. This light microscopic somatic spines regress, and basket axons form normal evidence does not exclude the possibility that GAD- pericellular baskets and pinceau formations. The eliminaimmunonegative baskets exist, although previous electron tion of basket cells may be achieved by appropriately timed microscopic observations did not reveal such structures X-irradiation (Altman, ’76). If the arrival of basket cell (Caddy and Biscoe, ’79; Heckroth et al., ’90). axons on the Purkinje cell somata was a motivating factor The cellular origin of the GAD-IR puncta observed in in climbing fiber ascent, then, in the absence of basket cells, association with Purkinje cell somata in lurcher cannot be numerous long pedunculated spines would be formed on proven on the basis of the present data. Such elements the principal dendritic trunks of the Purkinje cells, and could arise from GAD-IR neurons other than basket cells, somatic spines would be retained. Neither of these abnormalities is described by Altman, and the illustrations provided clearly indicate that this lack of description is no oversight. Fig. 7. GAD-IR elements in P15 normal (A,C, and E)and lurcher Thus it appears to be a reasonable assertion that, in the (B,D, and F) mice, as observed with the inclusion of Triton X-100 in the absence of basket cells, climbing fibers indeed ascend the incubation media. Note reduced cortical thickness in lurcher at this age Purkinje cell dendrites. The formation of inhibitory pericel(B). GAD-IR fibers (arrowheads) completely surround Purkinje cell somata in normal cerebellum (C), but are concentrated around the lular baskets and pinceau formations, and the ascent of climbing fibers, each may proceed normally in the absence apical somata and primary dendrite in lurcher (D). Basal portions of lurcher Purkinje cell somata are free of GAD-IR profiles (open arrows in of the other. D). Pinceau formations are conspicuous GAD-IR structures in normal In lurcher, however, both systems fail. A parsimonious animals at this age (black arrows in C and El, but are not observed in interpretation of these data is that a defect in the common lurcher. Note the lurcher Purkinje cell axon in F (open arrow) that element, the Purkinje cell (known to be directly affected by crosses the granular layer without any GAD-IR investment. g, granular layer; gl, glomerulus; m, molecular layer; P, Purkinje cell; pc, Purkinje Lc), causes abnormal development of both afferent fiber types. Indeed, because of the exchange of position required cell layer. ~~

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for normal development of these afferents, the developmental arrest of only one of the fiber types could physically prevent normal maturation of the other. Whether one or the other afferent type is capable of normal maturation in lurcher could be determined through experimental elimination of olivocerebellar fibers, or of basket cells in lurcher, as has been achieved in normal animals. Such manipulations would remove any physical hinderance of one fiber type by the other, and so reveal whether the development of one or both of these Purkinje cell afferents is directly effected by Lc. It is of interest to consider the functional consequences of the rearrangement of synaptic inputs to the Purkinje cells observed in lurcher, in terms of the physiology and viability of Purkinje cells. Although morphological alterations of lurcher Purkinje cells are already present in the early postnatal period, their degeneration apparently does not begin until P8-P10 (Caddy and Biscoe, ’79), the same ages when both climbing fibers and basket cell axons begin to exhibit abnormalities in their development. Perhaps the inversion of inhibitory and excitatory inputs on the Purkinje cell surface which occurs in lurcher, in contrast to simply the absence of one or the other afferent fiber (Altman, ‘76; Sotelo and Arsenio-Nunes, ’76), is a lethal situation for the Purkinje cells. Thus, for instance, an intrinsic defect in Purkinje cell dendrites preventing climbing fiber ascent would compound to produce anomalies in both climbing fiber and basket cell axon development, and finally Purkinje cell death as a result of inappropriate spatial distribution of excitatory and inhibitory synaptic activity. The effects of the abnormal placement of inputs on neurophysiological parameters and on intracellular signaling and metabolism in lurcher Purkinje cells have yet to be examined. In summary, examination of GAD-IR elements in the cerebellum of developing lurcher and normal mice reveals the presence of caps around the upper portion of Purkinje cell somata in lurcher, rather than the typical baskets and pinceau formations found in the normal cerebellum. In addition, two early phenotypic expressions of the lurcher gene have been described: (1)the reduced complexity of the Purkinje cell axonal plexus in the granular layer, as early as P3, and (2) the presence of GAD-IR processes within the lurcher EGL throughout the early postnatal period.

ACKNOWLEDGMENTS The author is indebted to Ms. Vickey S. Summers, HT-ASCP, for her expert technical assistance. This project was supported by grant 5 SO7 RR5371, awarded by the Biomedical Research Support Grant Program, Division of Research Resources, National Institutes of Health.

LITERATURE CITED Altman, J. (1976) Experimental reorganization of the cerebellar cortex. V. Effects of early X-irradiation schedules that allow or prevent the acquisition ofbasket cells. J. Comp. Neurol. 1 6 5 3 - 4 8 . Batini, C., C. Buisseret-Delmas, C. Compoint, and H. Daniel (1989) The GABAergic neurons of the cerebellar nuclei in the rat: Projections to the cerebellar cortex. Neurosci. Lett. 99251-256. Biscoe, T.J., J.P. Fry, C. Rickets, and J.-Y. Wu (1984) Immunocytochemical visualization of L-glutamate decarboxylase in the cerebellum of the normal and the mutant mouse Lurcher. J. Physiol. (Lond.) 353:122P. Caddy, K.W.T., and T.J. Biscoe (1975) Preliminary observations on the cerebellum in the mutant mouse Lurcher. Brain Res. 912276-280.

Caddy, K.W.T., and T.J. Biscoe (1979) Structural and quantitative studies on the normal C3H and lurcher mutant mouse. Phil. Trans. Roy. SOC. London 287:167-201. Chen, S., and D.E. Hillman (1989) Regulation of granule cell number by a predetermined number of Purkinje cells in development. Dev. Brairi Res. 45: 137-147. Coyle, J.T., and S.J. Enna (1976) Neurochemical aspects of the ontogenesis of GABAergic neurons in the rat brain. Brain Res. 111:119-133. Das, G.D., and M.J. Pfaffenroth (1977) Experimental studies on the postnatal development of the brain. 111. Cerebellar development following localized administration of ENU. Neuropath. Appl. Neurobiol. 3:191212. Fortier, P.A., A.M. Smith, and S.Rossignol (1987) Locomotor deficits in the mutant mouse, Lurcher. Exp. Brain Res. 66.271-286. Fournet, N., L.M. Garcia-Segura, A.W. Norman, and L. Orci (1986) Selective localization of calcium-binding protein in human brainstem, cerebellum and spinal cord. Brain Res. 399:310-316. Fry, J.P., C. Rickets, and T.J. Biscoe (1985) On the location of gammaaminobutyrate and benzodiazepine receptors in the cerebellum of the normal C3H and Lurcher mutant mouse. Neuroscience 14:1091-1101. Garcia-Segura, L.M., D. Baetens, J. Roth, A.W. Norman, and L. Orci (1984) Immunohistochemical mapping of calcium-bindingprotein immunoreactivity in the rat central nervous system. Brain Res. 296:75-86. Hadjian, R.A., and J.A. Stewart (1977) Immunological quantitation of glutamic acid decarboxylase in developing mouse brain. J. Neurochem. 28:1249-1257. Heckroth, J.A., D. Goldowitz, and L.M. Eisenman (1990) Olivocerebellar fiber maturation in normal and lurcher mutant mice: Defective development in lurcher. J. Comp. Neurol. 291t415-430. Hendelman, W.J., and A S . Aggerwal(1980) The Purkinje neuron: I. A Golgi study of its development in the mouse and in culture. J. Comp. Neurol. 193: 1063-1079. Herrup, K., and R.J. Mullen (1979) Staggerer chimeras: Intrinsic nature of Purkinje cell defects and implications for normal cerebellar development. Brain Res. 178:443-457. Herrup, K., and K. Sunter (1987) Numerical matching during cerebellar development: Quantitative analysis of granule cell death in staggerer mouse chimeras. J. Neurosci. 72329-836. Landis, D.M.D., and R.L. Sidman (1978) Electron microscopic analysis; of postnatal histogenesis in the cerebellar cortex of staggerer mutant mice. J. Comp. Neurol. 179331-864. Mallet, J., M. Huchet, R. Pougeois, and J.-P. Changew (1976) Anatomical, physiological and biochemical studies on the cerebellum from mutant mice. 111. Protein differences associated with the weaver, staggerer, and nervous mutations. Brain Res. 103t291-312. Mariani, J., F. Crepel, K. Mikoshiba, J.P. Cbangeux, and C. Sotelo (19‘77) Anatomical, physiological and biochemical studies of the cerebellum London 281:1-28. from reeler mutant mouse. Phil. Trans. Roy. SOC. Meller, K., and P. Glees (1969) The development of the mouse cerebellum. A Golgi and electron microscopical study. In R. Llinas (ed): Neurobiology of Cerebellar Evolution and Development. Chicago: American Medical Association Education and Development, pp. 783-801. Norman, D.J., C. Fletcher, and N. Heintz (1991) Genetic mapping of the lurcher locus on mouse chromosome 6 using an intersubspecific backcross. Genomics 9:147-153. Oertel, W.H., E. Mugnaini, D.E. Schmechel, M.L. Tappaz, and I.J. Kopin (1982) The immunocytochemical demonstration of gamma-aminobutyric acid-ergic neurons-methods and application. In V. Chan-Palay and S.L. Palay (eds): Cytochemical Methods in Neuroanatomy. New York: Alan R. Liss, pp. 297-329. Oertel, W.H., D.E. Schmechel, E. Mugnaini, M.L. Tappaz, and I.J. Kopin (1981)Immunocytochemical localization of glutamate decarboxylase m rat cerebellum with a new antiserum. Neuroscience 6:2715-2735. Phillips, R.J.S. (1960) “Lurcher”, a new gene in linkage group XI of the house mouse. J. Genet. 57:35-42. Roth, J., D. Baetens, A.W. Norman, and L.M. Garcia-Segura (1981) Specific neurons in chick central nervous system stain with an antibody against chick intestinal vitamin D-dependent calcium-binding protein. Brain Res. 222:452457. Saito, K., R. Barber, J.Y. Wu, T. Matsuda, E. Roberts, and J. Vaughn (1974) Immunohistochemical localization of glutamate decarboxylase in rat cerebellum. Proc. Nat. Acad. Sci. U.S.A. 71.269-273. Sotelo, C. (1990) Axonal abnormalities in cerebellar Purkinje cells of the “hyperspiny Purkinje cell” mutant mouse. J. Neurocytol. 19:737-755.

GAD-IMMUNOREACTIVITY IN LURCHER CEREBELLAR CORTEX Sotelo, C., and M.L. Arsenio-Nunes (1976)Development of Purkinje cells in absence of climbing fibers. Brain Res. 111:389-395. Sotelo, C., and A. Triller (1979) Fate of presynaptic d e r e n t s to Purkinje cells in the adult nervous mutant mouse: A model to study presynaptic stabilization. Brain Res. 17511-36. Swisher, D.A., and D.B. Wilson (1977) Cerebellar histogenesis in the lurcher (Lc) mutant mouse. J. Comp. Neurol. 173.205-218. Unsworth, B.R., L.H. Fleming, and P.C. Caron (1980) Neurotransmitter enzymes in telencephalon, brain stem, and cerebellum during the entire life span of the mouse. Mech. Ageing Dev. 13:205-217. Vogel, M.W., and K. Herrup (1989) Numerical matching in the mammalian CNS: Lack of a competitive advantage of early over late-generated cerebellar granule cells. J.Comp. Neurol. 283:118-128. Wassef, M., J. Simons, M.L. Tappaz, and C. Sotelo (1986) Non-F’urkinje cell GABAergic innervation of the deep cerebellar nuclei: A quantitative immunocytochemical study in C57BL and in Purkinje cell degeneration mutant mice. Brain Res. 399:125-135.

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Wetts, R., and K. Herrup (1982a) Interaction of granule, Purkinje and inferior olivary neurons in lurcher chimeric mice. 11. Granule cell death. Brain Res. 250:358-362. Wetts, R., and K. Herrup (1982b) Interaction of granule, Purkinje and inferior olivary neurons in Lurcher chimaeric mice. I. Qualitative studies. J. Embryol. Exp. Morphol. 68r87-98. Wetts, R., and K. Herrup (1983) Direct correlation between Purkinje cell number in the cerebella of lurcher chimeras and wild-type mice. Dev. Brain Res. 10:41-47. Wilson, D.B. (1975) Brain abnormalities in the lurcher (Lc) mutant mouse. Experientia 31.220-221. Wilson, D.B. (1976) Histological defects in the cerebellum of adult lurcher (lc) mice. J. Neuropath. Exp. Neurol. 35:40-45. Wu, J.-Y., C.T. Lin, C. Brandon, T.-S.Chan, H. Mohler, and J.G. Richards (1982) Regulation and immunocytochemical characterization of glutamic acid decarboxylase. In V. Chan-Palay and S.L. Palay (eds): Cytochemical Methods in Neuroanatomy. New York Alan R. Liss, pp. 279-296.