Immunological studies on the Purkinje cells from rat and mouse cerebella. I. Evidence for antibodies characteristic of the Purkinje cells.

Antibodies have been raised against an enriched preparation of isolated rat cerebellar Purkinje cells. The immunoglobulins were labeled with ¹²⁵I and the strength and specificity of the serum determined by a direct binding assay on cerebellar membranes. About 2% of the ¹²⁵I-labeled IgG bound to an excess of cerebellar membranes. Absorption with heart and liver membranes removed 80.5% of the ¹²⁵I-labeled IgG binding to cerebellar membranes; absorption with cerebrum membranes removed 13% more; the remaining 6.5% were directed specifically against cerebellar membranes. An enriched ¹²⁵I-labeled anti-Purkinje antibody population was prepared by absorption and subsequent elution from cerebellar membranes. The absorption pattern with heart, liver, and cerebrum membranes resembled that found with the total population of IgG except that the nonspecific binding was significantly diminished. The Purkinje cell degeneration (pcd) mouse mutant was used to assess the specificity of the serum toward the Purkinje cells. After absorption of the enriched anti-Purkinje antibodies with heart, liver, and cerebrum membranes, the binding of labeled IgG to membranes prepared from pcd/pcd cerebella was about one-fourth that found with control cerebella. The direct immunoperoxidase technique performed on smears of purified Purkinje and granule cells shows that the unabsorbed serum stains both classes of cells, but that after absorption with liver, heart, and cerebrum membranes, only the Purkinje cells react positively.     

GABA and synaptic inhibition of mouse cerebellum lacking glutamate decarboxylase 67

γ-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter and also presumed to be a neurotrophic factor. GABA is synthesized by glutamate decarboxylase (GAD). A mouse lacking a 67 kDa isoform of GAD (GAD67) has a reduced GABA level in its brain at birth and does not survive postnatally because of cleft palate. In this study, to investigate the functional and developmental roles of GABA in the postnatal cerebellum, selective GAD67 deletion was achieved using a Cre-loxP strategy. In this mouse, GABA level was reduced to 16-44% in the cerebellum but not in the cerebrum. Inhibitory synaptic transmission to Purkinje cells was seriously impaired. However, the morphology of Purkinje cells and the density of synaptic terminals in the cerebellar cortex appeared unaffected, suggesting that GABA does not participate in cerebellar development substantially.     

The abnormal cerebellar organization of Weaver and reeler mice does not affect the cellular distribution of three neuronal mRNAs

We used in situ hybridization of 35S-labeled antisense RNAs to study the cellular distribution of three neuronal mRNAs. We compared the expression of these RNAs in cerebellar Purkinje neurons in wild-type (C57Bl-6J) mice and in two mutants (Weaver and reeler) known to have abnormal cerebellar morphologies. In normal mice, GAD mRNA is present in four sets of neurons in the cerebellar cortex while calbindin mRNA is present only in Purkinje neurons. Proenkephalin mRNA is present in Golgi II neurons as well as in a set of neurons in the deep part of the molecular layer. Despite the dramatic differences in structural organization and inputs of Purkinje neurons in the cerebella of adult Weaver and reeler mice, the expression of these RNAs appears unchanged. These results support the hypothesis that Purkinje cell cytodifferentiation proceeds autonomously after its inception in early embryonic life.     

Levels of glutamate,aspartate, GABA,and taurine in different regions of the cerebellum after X-irradiation-induced neuronal loss

The levels of glutamate (Glu), aspartate (Asp), -amino-n-butyric acid (GABA), and taurine (Tau) were determined in the cortex, molecular layer, and deep nuclei of cerebella of adult rats exposed to X-irradiation at 12–15 days following birth (to prevent the acquisition of late-forming granule cells; 12–15x group) and 8–15 days following birth (to prevent the acquisition of granule and stellate cells; 8–15x group). Also, the levels of the four amino acids were measured in the crude synaptosomal fraction (P₂) isolated from the whole cerebella of the control, 12–15x, and 8–15x groups. The level of Glu was significantly decreased by (1) 6–20% in the cerebellar cortex; (2) 15–20% in the molecular layer; and (3) 25–50% in the P₂ fraction of the X-irradiated groups relative to control values. The content of Glu in the deep nuclei was not changed by X-irradiation treatment. Regional levels of Asp were unchanged by X-irradiation, while its level in P₂ decreased by 15–30% after treatment. The levels of GABA and Tau in the molecular layer, deep nuclei, or P₂ were not changed in the experimental groups. However, there was a 15% increase in the levels of GABA and Tau in the cerebellar cortex of the 8–15x group relative to control values. The data support the proposed role of glutamate as the excitatory transmitter released from the cerebellar granule cells but are inconclusive regarding a transmitter role for either Tau or GABA from cerebellar stellate cells.     

Hypoxic Preconditioning and Hypoxic-Ischemic Brain Damage in the Immature Rat: Pathologic and Metabolic Correlates

Abstract: It has been reported that immature rats subjected to cerebral hypoxia-ischemia sustain less brain damage if they are previously exposed to systemic hypoxia compared with animals not exposed to prior hypoxia. Accordingly, neuropathologic and metabolic experiments were conducted to confirm and extend the observation that hypoxic preconditioning protects the perinatal brain from subsequent hypoxic-ischemic brain damage. Six-day postnatal rats were subjected to systemic hypoxia with 8% oxygen at 37°C for 2.5 h. Twenty-four hours later, they were exposed to unilateral cerebral hypoxia-ischemia for 2.5 h, produced by unilateral common carotid artery ligation and systemic hypoxia with 8% oxygen. Neuropathologic analysis, conducted at 30 days of postnatal age, indicated a substantial reduction in the severity of brain damage in the preconditioned rats, such that only 6 of 14 such animals exhibited cystic infarction, but all 13 animals without prior preconditioning exhibited infarction ( p < 0.001). Measurement of cerebral glycolytic and tricarboxylic acid intermediates and high-energy phosphate reserves at the terminus of and at 4 and 24 h following hypoxia-ischemia showed no differences in the extent of alterations in the preconditioned and nonpreconditioned immature rats. A difference was seen in the restitution of high-energy stores during the first 24 h of recovery from hypoxia-ischemia, with a more optimal preservation of these metabolites in the preconditioned animals, reflecting the less severe ultimate brain damage. Accordingly, the neuroprotection afforded to the preconditioned animals was not the result of any differences in the extent of anaerobic glycolysis, tissue acidosis, or depletion in high-energy reserves during hypoxia-ischemia but rather the result of other mechanisms that improved the metabolic status of the immature brain during the early hours of reperfusion following hypoxia-ischemia.     

Cerebellar plasma membrane proteins and their antisera

Plasma membranes have been isolated from neonatal through adult cerebella by a sequence of differential centrifugation, aqueous two-phase polymer fractionation and density gradient centrifugation. The protein composition of cerebellar membranes from various aged mice was compared by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Increases in the relative amount of membrane proteins with molecular weights (X 10(-3)) of 400, 340, 270, 220, 54, 44, and 9.5 were most pronounced, while a protein of 66,000 Mr disappeared between birth and Day 25. The relationship of these proteins and others to specific cell types in the cerebellum was examined by preparing membrane fractions from isolated granule and Purkinje cells, as well as from the cerebella of neurological mutant mice: reeler, weaver, staggerer, and nervous. In addition, those membrane proteins on the surface of dissociated cerebellar cells were identified by lactoperoxidase-catalyzed iodination, while glycoproteins were identified by galactose oxidase treatment and NaB3H4 reduction. Rabbit antisera were prepared toward those SDS-PAGE membrane proteins which appeared cell specific or developmentally regulated. Sera from these rabbits were used with indirect immunoperoxidase and immunofluorescence to stain frozen sections of mouse cerebellum and dissociated cerebellar cell cultures. In tissue sections antiserum toward the 400,000 Mr protein (p400) and antiserum p14.7 gave strong reactions with Purkinje cells while anti-p130 reacted preferentially with Purkinje cell somas, anti-p220 stained small cells in the internal granule layer and anti-p30 displayed a coarse, grainy staining of the granule and molecular layers, characteristic of a synaptic localization. Only anti-p220 and anti-p130 bound to freshly dissociated cells or cultured cerebellar cells. Large phase-bright cells in the cultures bound antiserum p130. Anti-p220 reacted specifically with a subpopulation of small round viable cells that bound tetanus toxin and decreased in number from 9% at Day 3 to 0.5% of the cells by Day 11, suggestive of granule neurons.     

Comparison of the effects of barbiturate and ethanol given to neonates on the cerebellar morphology

Previous studies from different laboratories have suggested that neonatal exposure to barbiturate and ethanol induces long-term changes in cerebellar morphology. The present study was designed to compare in similar conditions the effect of neonatal exposure to maximal doses of barbiturate or ethanol on cerebellar morphology. Phenobarbital was administered via daily injections of 50 mg/kg on neonatal days 2-21 (B group). Ethanol was similarly administered in doses of 3 g/kg (E3g) and the submaximal dose of 2 g/kg (E2g). At age 50 days, the cerebella of treated and control offspring were subjected to histological analysis. The sagittal areas of the cerebellar layers were similarly reduced compared to controls in both B and E3g groups. In addition, B and E3g groups exhibited a similar deficit in the number of the cerebellar Purkinje and granule neurons. As barbiturate and E3g, a submaximal dose of ethanol (B2g) induced a deficit in the number of cerebellar Purkinje cells. However, it did not affect the granule cells and the area of the cerebellar layers. The results suggest that under standardized conditions, barbiturate and ethanol have a similar potent neurotoxic effect on the cerebellum. That is, they both impair the development of the cerebellar layers to a similar extent and destroy neurons even after they have already formed.     

The Immp2l mutation causes age‐dependent degeneration of cerebellar granule neurons prevented by antioxidant treatment

Reactive oxygen species are implicated in age‐associated neurodegeneration, although direct in vivo evidence is lacking. We recently showed that mice with a mutation in the Inner Mitochondrial Membrane Peptidase 2‐like (Immp2l) gene had elevated levels of mitochondrial superoxide, impaired fertility and age‐associated phenotypes, including kyphosis and ataxia. Here we show that ataxia and cerebellar hypoplasia occur in old mutant mice (> 16 months). Cerebellar granule neurons (CGNs) are significantly underrepresented; Purkinje cells and cells in the molecular layer are not affected. Treating mutant mice with the mitochondria‐targeted antioxidant SkQ1 from 6 weeks to 21 months protected cerebellar granule neurons. Apoptotic granule neurons were observed in mutant mice but not in age‐matched normal control mice or SkQ1‐treated mice. Old mutant mice showed increased serum protein carbonyl content, cerebellar 4‐hydroxynonenal (HNE), and nitrotyrosine modification compared to old normal control mice. SOD2 expression was increased in Purkinje cells but decreased in granule neurons of old mutant mice. Mitochondrial marker protein VDAC1 also was decreased in CGNs of old mutant mice, suggesting decreased mitochondrial number. SkQ1 treatment decreased HNE and nitrotyrosine modification, and restored SOD2 and VDAC1 expression in CGNs of old mutant mice. Neuronal expression of nitric oxide synthase was increased in cerebella of young mutant mice but decreased in old mutant mice. Our work provides evidence for a causal role of oxidative stress in neurodegeneration of Immp2l mutant mice. The Immp2l mutant mouse model could be valuable in elucidating the role of oxidative stress in age‐associated neurodegeneration.     

Ectopic Purkinje-like cells are GABAergic: Immunohistochemistry with an immune serum against glutamic acid decarboxylase

Summary Intensely stained cells are found in the cerebellar white matter of the vermis and paravermis in adult rats after immunoreaction with an immune serum raised against glutamic acid decarboxylase (GAD). The cells are similar in size to cortical Purkinje cells and three times the size of Golgi cells of the internal granule layer, and have a thick immunopositive cell process emerging from a welldefined cytoplasmic cone. In the cytoplasm, immunoprecipitates are more dense around the nucleus as in normally located Purkinje cells. The morphological appearance of the immunopositive cells suggests that they may be ectopically located Purkinje cells. The soma of the ectopic Purkinje cells is contacted by a few darkly stained terminal boutons. Data indicate that, in spite of the different cellular environment, ectopic Purkinje cells can develop not only the typical morphological pattern already described but also other intrinsic features, such as their typical inhibitory neurotransmitter.     

Ascorbic Acid Ameliorates Gestational Lead Exposure-Induced Developmental Alteration in GAD67 and c-Kit Expression in the Rat Cerebellar Cortex

In the present study, we investigated the effects of ascorbic acid on lead-exposed developing cerebellum. Female rats were divided into the following three groups: control (distilled water), lead (0.2% lead acetate), and lead plus ascorbic acid (100 mg/kg/day, 10% solution). To evaluate the effect of lead exposure and ascorbic acid treatment accurately on the cerebellar development for the gestational period, we halted further treatment with lead and ascorbic acid in the dams after delivery of the pups. Although the ascorbic acid slightly decreased the lead level in pups, lead level was still high in the group treated with lead plus ascorbic acid group compared with the control group. The blood lead levels indicated that the ascorbic acid could facilitate both the excretion and transfer of lead from a dam to its pups via milk. At postnatal day 21, lead exposure significantly reduced the number of Purkinje cells in the cerebellar cortex of pups. Additionally, lead treatment induced degenerative changes such as reduction of glutamic acid decarboxylase (GAD67) and c-kit expressions are observed in the developing cerebellar cortex. In the cerebellum of the pups from the lead plus ascorbic acid group, reduction of the number of Purkinje cells, GAD67 expression, and c-kit immunopositivity were remarkably restored compared with the lead group. Our present results suggested that ascorbic acid treatment to lead-exposed dam exerted protective effects on the developing cerebellum against lead-induced neurotoxicity.     

Growth arrest specific gene 1 is a positive growth regulator for the cerebellum.

Postnatal cerebellum development involves the generation of granule cells and Bergmann glias (BGs). The granule cell precursors are located in the external germinal layer (EGL) and the BG precursors are located in the Purkinje layer (PL). BGs extend their glial fibers into the EGL and facilitate granule cells' inward migration to their final location. Growth arrest specific gene 1 (Gas1) has been implicated in inhibiting cell-cycle progression in cell culture studies (G. Del Sal et al., 1992, Cell 70, 595--607). However, its growth regulatory function in the CNS has not been described. To investigate its role in cerebellar growth, we analyzed the Gas1 mutant mice. At birth, wild-type and mutant mice have cerebella of similar size; however, mature mutant cerebella are less than half the size of wild-type cerebella. Molecular and cellular examinations indicate that Gas1 mutant cerebella have a reduced number of granule cells and BG fibers. We provide direct evidence that Gas1 is required for normal levels of proliferation in the EGL and the PL, but not for their differentiation. Furthermore, we show that Gas1 is specifically and coordinately expressed in both the EGL and the BGs postnatally. These results support Gas1 as a common genetic component in coordinating EGL cell and BG cell proliferation, a link which has not been previously appreciated.     

Preconditioning Triggered by Carbon Monoxide (CO) Provides Neuronal Protection Following Perinatal Hypoxia-Ischemia

Perinatal hypoxia-ischemia is a major cause of acute mortality in newborns and cognitive and motor impairments in children. Cerebral hypoxia-ischemia leads to excitotoxicity and necrotic and apoptotic cell death, in which mitochondria play a major role. Increased resistance against major damage can be achieved by preconditioning triggered by subtle insults. CO, a toxic molecule that is also generated endogenously, may have a role in preconditioning as low doses can protect against inflammation and apoptosis. In this study, the role of CO-induced preconditioning on neurons was addressed in vitro and in vivo. The effect of 1 h of CO treatment on neuronal death (plasmatic membrane permeabilization and chromatin condensation) and bcl-2 expression was studied in cerebellar granule cells undergoing to glutamate-induced apoptosis. CO's role was studied in vivo in the Rice-Vannucci model of neonatal hypoxia-ischemia (common carotid artery ligature +75 min at 8% oxygen). Apoptotic cells, assessed by Nissl staining were counted with a stereological approach and cleaved caspase 3-positive profiles in the hippocampus were assessed. Apoptotic hallmarks were analyzed in hippocampal extracts by Western Blot. CO inhibited excitotoxicity-induced cell death and increased Bcl-2 mRNA in primary cultures of neurons. In vivo, CO prevented hypoxia-ischemia induced apoptosis in the hippocampus, limited cytochrome c released from mitochondria and reduced activation of caspase-3. Still, Bcl-2 protein levels were higher in hippocampus of CO pre-treated rat pups. Our results show that CO preconditioning elicits a molecular cascade that limits neuronal apoptosis. This could represent an innovative therapeutic strategy for high-risk cerebral hypoxia-ischemia patients, in particular neonates.     

Feulgen-DNA content of the Purkinje neuron: "diploid" or "tetraploid"?

Microdensitometric measurements of the Feulgen-DNA content of the Purkinje cells and of the small granular cells (2c control) were carried out at lambda 550 nm and by the two wavelength method according to Fukuda et al., and were also corrected for the glare. We analysed sections of perfused cerebella and isolated cells of cerebellar cortex from adult rats. The Purkinje cells had a mean value of Feulgen-DNA content 30-45% higher than the 2c value, irrespective of the methods of preparation or measurement.     

Postnatal expression of glutamate decarboxylases in developing rat cerebellum

The recent identification of two genes encoding distinct forms of the GABA synthetic enzyme, glutamate decarboxylase (GAD), raises the possibility that varying expression of the two genes may contribute to the regulation of GABA production in individual neurons. We investigated the postnatal development the two forms of GAD in the rat cerebellum. The mRNA for GAD₆₇, the form which is less dependent on the presence of the cofactor, pyridoxal phosphate (PLP), is present at birth in presumptive Purkinje cells and increases during postnatal development. GAD₆₇ mRNA predominates in the cerebellum. The mRNA for GAD₆₅, which displays marked PLP-dependence for enzyme activity, cannot be detected in cerebellar cortex by in situ hybridization until P7 in Purkinje cells, and later in other GABA neurons. In deep cerebellar nuclei, which mature prenatally, both forms of GAD mRNA can be detected at birth. The amounts of immunoreactice GAD and GAD enzyme activity parallel changes in mRNA levels. We suggest that the delayed appearance of GAD₆₅ is coincident with synapse formation between GABA neurons and their targets during the second postnatal week. GAD₆₇ mRNA may be present prior to synaptogenesis to produce GABA for trophic and metabolic functions.Special issue dedicated to Dr. Eugene Roberts.     

Comparative aspects of cerebellar organization. From mormyrids to mammals.

Recent progress in the comparative analysis of the vertebrate cerebellar organization shows that the cerebella of different tetrapods have a basically similar intrinsic organization, whereas the cerebellum of fishes displays a number of fundamental differences in this respect. Clear examples of teleostean cerebellar specializations are present in the gigantocerebellum of mormyrids, including a valvula cerebelli, the absence of a parasagittal zonal organization, the presence of eurydendroid projection neurons instead of deep cerebellar nuclei, a precerebellar nucleus lateralis valvulae, olivocerebellar fibers that do not climb into the molecular layer, uni- and bilateral locations of granule cells, parallel fibers without a T-shaped bifurcation and with a coextensive distribution in the transverse plane, and different Purkinje cell arrangements including a dendritic palisade pattern. A theoretical exploration of the possible significance of these configurations suggests that they all might be involved in a single main cerebellar function, i.e. coincidence detection of parallel fiber activity by Purkinje cells.     

Effect of bax deletion on ethanol sensitivity in the neonatal rat cerebellum

The developing cerebellum is highly sensitive to ethanol during discrete neonatal periods. This sensitivity has been linked to ethanol-induced alterations in molecules of the Bcl-2 survival-regulatory gene family. Ethanol exposure during peak periods of cerebellar sensitivity, for example, results in increased expression of proapoptotic proteins of this family, while overexpression of the antiapoptotic Bcl-2 protein in the nervous system protects against ethanol neurotoxicity. For the present study, neonatal mice with a targeted deletion of the proapoptotic bax gene were used to determine whether elimination of this protein would mitigate ethanol toxicity. bax knock-out and wild-type mice pups were exposed to ethanol via vapor inhalation during the maximal period of neonatal cerebellar ethanol sensitivity and cerebellar tissue was subsequently assessed for Purkinje and granule cell number and ethanol-mediated generation of reactive oxygen species (ROS). The results revealed that: (1) ethanol exposure during the peak period of cerebellar vulnerability resulted in substantial loss of Purkinje cells in wild-type animals, but not in bax knock-outs; (2) granule cells in the bax gene-deleted animals were not similarly protected from ethanol effects; and (3) levels of ROS following acute ethanol exposure were appreciably enhanced in the wild-type animals but not in the bax knock-outs. These results imply that Bax is important to ethanol-induced Purkinje cell death during critical neonatal periods, but that ethanol effects on granule cells may function at least partially independent of this apoptosis agonist. Amelioration of ethanol-mediated increases in ROS production in the knock-outs may contribute to the observed effects.     

ATP-Dependent Glutamate Uptake into Synaptic Vesicles from Cerebellar Mutant Mice

The ATP-dependent glutamate uptake system in synaptic vesicles prepared from mouse cerebellum was characterized, and the levels of glutamate uptake were investigated in the cerebellar mutant mice, staggerer and weaver, whose main defect is the loss of cerebellar granule cells, and the nervous mutant, whose main defect is the loss of Purkinje cells. The ATP-dependent glutamate uptake is stimulated by low concentrations of chloride, is insensitive to aspartate, and is inhibited by agents known to dissipate the electrochemical proton gradient. These properties are similar to those of the glutamate uptake system observed in the highly purified synaptic vesicles prepared from bovine cortex. The ATP-dependent glutamate uptake system is reduced by 68% in the staggerer and 57-67% in the weaver mutant; these reductions parallel the substantial loss of granule cells in those mutants. In contrast, the cerebellar levels of glutamate uptake are not altered significantly in the nervous mutant, which has lost Purkinje cells, but not granule cells. In view of evidence that granule cells are glutamatergic neurons and Purkinje cells are GABAergic neurons, these observations support the notion that the ATP-dependent glutamate uptake system is present in synaptic vesicles of glutamatergic neurons.     

Effects of selective doses of x-irradiation on the levels of several amino acids in the cerebellum of the rat

The cerebella of rats were exposed to selective doses of low levels of x-irradiation beginning on day 4, 8, or 12 following birth. The doses of x-irradiation given on days 12, 13, and 15 (12–15X group) resulted in a 24% reduction in the wet weight of the cerebella; the doses given on days 8, 9, 11, 13, and 15 (8–15X group) resulted in a 57% weight reduction; the doses given on days 4, 5, 7, 9, 11, 13, and 15 (4–15X group) resulted in a 67% weight reduction. The schedule of x-irradiation begun on day 12, which prevented the acquisition of the late-forming granule cells, reduced the levels (nmole/mg dry tissue weight) of alanine (22%) and glutamate (10%), and increased the levels of glycine (15%), GABA (13%), and taurine (71%), with respect to control values. The schedule begun on day 8, which prevented the acquisition of stellate and granule cells, reduced the levels of alanine (15%), glutamate (12%), and taurine (21%), and increased the levels of glycine (102%) and GABA (56%). The schedule begun on day 4, which prevented the acquisition of basket, stellate, and granule cells, reduced the level of glutamate (15%) and increased the levels of glycine (186%) and GABA (78%). The levels of alanine and taurine in the cerebella of the 4–15X group were the same as control values. The level of aspartate in the cerebella of the 3 groups of x-irradiated animals was not significantly different from control values. The consistent reduction in the level of glutamate as a function of the number of doses of x-irradiation is suggestive that glutamate may have a higher level in the granule cells than in other cells in the cerebellum, and that the higher level may be a reflection of a possible excitatory transmitter role for glutamate. In addition, the data are interpreted in terms of taurine being associated with the stellate cells and possibly serving as a transmitter for these inhibitory interneurons.     

Cyclic Nucleotide-Dependent Protein Kinases and Some Major Substrates in the Rat Cerebellum After Neonatal X-Irradiation

The levels of cAMP-dependent protein kinase (type I), or cGMP-dependent protein kinase, or protein I, and of a 23,000 MW substrate for the cGMP-dependent protein kinase were measured in cerebella from normal rats and in the cerebella from rats in which a selective loss of interneurons in the cerebellar cortex had been produced by X-irradiation. A decrease was observed in the concentrations of cAMP-dependent protein kinase and of protein I, whereas an increase was observed in the concentrations of cGMP-dependent protein kinase and of the 23,000 MW substrate. The data, taken together with the results of other studies, support the interpretation that cAMP-dependent protein kinase and protein I are distributed throughout the cerebellum, but that cGMP-dependent protein kinase and the 23,000 MW substrate are highly concentrated in Purkinje cells.