Surviving Purkinje Cells Research Articles

Granule Neuron Regulation of Purkinje Cell Development: Striking a Balance Between Neurotrophin and Glutamate Signaling

Granule neurons, presynaptic afferents of Purkinje cells, are potent regulators of Purkinje cell development. Purified Purkinje cells survive and differentiate poorly, whereas coculture with granule neurons enhances their survival and dendritic development. Here we investigate the role of neurotrophins in granule-Purkinje cell interactions. BDNF or NT-4 improves, but NT-3 or CNTF reduces, survival of isolated Purkinje cells. When granule neurons are present, however, BDNF or NT-4 treatment leads to Purkinje cell loss. This decrease is overcome by anti-BDNF or TrkB-IgG-blocking reagents or by CNQX, a non-NMDA glutamate receptor antagonist. Furthermore, BDNF increases the spine density on the surviving Purkinje cells. These results suggest that Purkinje cell survival and differentiation are context-dependent and require a balance between neurotrophin- and activity-dependent signaling.

Images in neuroscience. Brain development, II. Establishing synaptic connections.

Back to table of contents Previous article Next article Images in NeuroscienceFull AccessBrain Development, II: Establishing Synaptic ConnectionsCarol A. Tamminga, M.D., EditorCarol A. TammingaSearch for more papers by this author, M.D., EditorPublished Online:1 May 1998https://doi.org/10.1176/ajp.155.5.581AboutSectionsView articleView PDFView EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InEmail View articleDuring normal CNS development, neurons in various brain regions express chemical markers that attract ingrowing neuronal terminals to form synapses with their correct targets. A single group of neurons within a neuronal structure may offer distinctive chemical cues that are organized into meaningful anatomic patterns to attract afferents from different brain regions. The cerebellum, for example, receives axons from spinal cord neurons whose terminals are distributed into distinct columns in the cerebellar cortex (bright pink areas in left figure). The terminals of spinocerebellar axons are labeled by injecting a dye, horseradish peroxidase, into the spinal cord, where it is taken up by neurons and transported to their terminal ends. The distinctive distribution of terminals in the cerebellum correlates with biochemical differences among cerebellar Purkinje cells. Thus, it is thought that the Purkinje cells express the chemical markers that organize the spinocerebellar terminals into distinct fields.This model is being tested by studying a genetic strain of mice, staggerer, that fails to develop mature Purkinje cells. Staggerer (gene symbol, sg) is an autosomal recessive mutation that causes a block in the development of cerebellar Purkinje cells. The number of Purkinje cells is reduced by 75%, and the surviving Purkinje cells fail to differentiate completely. As a consequence of this Purkinje cell loss, the vast majority of granule cells (primary afferents to Purkinje cells) in the cerebellar cortex also die. If Purkinje cells are the organizers for cerebellar afferent projections, then in their absence the organization of spinocerebellar afferents should be disrupted. In older staggerer mice, the terminals of spinocerebellar axons are indeed reduced in density and lack any apparent organization. However, in juvenile staggerer mice (right figure), the afferent terminals still can be seen to form a pattern that is approximately normal, even in a cerebellum diminished by granule and Purkinje cell loss. The results suggest that even immature Purkinje cells may express the chemical markers that allow afferent axons to sort out into distinct columns. Modern molecular techniques have identified a growing number of transcription factors (DNA binding proteins such as Engrailed and Pax), nerve cell adhesion molecules (e.g., N-CAM), and signaling molecules (e.g., the semiphorins and collapsins) with distinct expression patterns in the developing nervous system. These molecules and the cells that express them are under intense study so that we can identify the molecular mechanisms that organize how the nervous system becomes wired together during development.Address reprint requests to Dr. Tamminga, Maryland Psychiatric Research Center, P.O. Box 21247, Baltimore, MD 21228. Photographs courtesy of Dr. Vogel. FIGURE 1. The horseradish-peroxidase-labeled terminals of spinocerebellar mossy fiber afferents (bright pink areas) segregate into discrete columns in the cerebellar cortex of wild type mice (left photomicrograph) and juvenile staggerer mutant mice (right photomicrograph), despite the defects in staggerer Purkinje cells and the loss of granule cells in this mutant. Scale bar, 200 µm. FiguresReferencesCited byDetailsCited ByNone Volume 155Issue 5 May 1998Pages 581-581 Metrics History Published online 1 May 1998 Published in print 1 May 1998

Reduced immunoreactivity to calcium-binding proteins in Purkinje cells precedes onset of ataxia in spinocerebellar ataxia-1 transgenic mice.

Earlier we have shown alterations in immunoreactivity (IR) to the calcium-binding proteins parvalbumin (PV) and calbindin D-28k (CaB) in surviving Purkinje cells of patients with spinocerebellar ataxia-1 (SCA-1). In the present study we determined PV and CaB expression (by immunohistochemical and immunoblot analyses) in Purkinje cells of transgenic mice (TM) expressing the human SCA-1 gene with an expanded (line B05) and normal (line A02) CAG tract, as well as in age-matched nontransgenic mice (nTM). Heterozygotes in the B05 line develop progressive ataxia beginning around 12 weeks of age. A02 animals are phenotypically indistinguishable from wild-type (nontransgenic) animals. In the cerebella of 8-, 9-, and 12-week-old TM-B05 there was a progressive decrease in PV IR in Purkinje cells compared with nTM and TM-A02. Parvalbumin immunostaining in interneurons was well preserved in all groups. A progressive decrease was also observed in CaB IR in Purkinje cells of 8-, 9-, and 12-week-old TM-B05. Cerebellar Purkinje cells of 6-week-old TM-B05, which exhibit no ataxia and even lack demonstrable Purkinje cell loss, also revealed reduction in PV IR. This change was matched by a significant decrease in the amount of cerebellar PV in 6-week-old TM-B05 as determined by Western blot analysis. Calbindin D-28K immunohistochemistry did not detect any marked changes in CaB IR within Purkinje cells at 4 weeks. However, at 6 weeks immunostaining and immunoblot analysis revealed a significant decrease in CaB in TM-B05 compared with controls. These data suggest that decreased levels of calcium-binding proteins in Purkinje cells in SCA-1 transgenic mice may cause alteration in Ca2+ homeostasis.

Functional NMDA Receptors Are Transiently Active and Support the Survival of Purkinje Cells in Culture

Conflicting evidence exists concerning the activity of NMDA receptors (NMDARs) in cerebellar Purkinje cells and their possible functions. To investigate the activity of NMDARS, we used whole-cell recording on immunocytochemically identified Purkinje cells in primary culture. In addition, we used mice with a disrupted NMDAR1 gene that lack functional NMDARs (NR1-/-) to assess the physiological role of NMDARs. In cultures from normal mice, NMDA-medicated currents were detected in all identified Purkinje cells at 4 d in vitro (div). After 14 d, however, NMDA responses were reduced in amplitude, whereas the responses to kainate and glutamate increased steadily in amplitude. In addition, the NMDA-induced current displayed a pronounced desensitization at these later stages; peak current declined to zero during steady application of NMDA. At 7 div, the number of surviving Purkinje cells was less in cultures treated with NMDA antagonists, and their survival was dose-dependent. Purkinje cell survival was correspondingly poorer in cultures from the NR1-/- mice than in wild-type controls, suggesting that NMDAR activity enhances the survival of Purkinje cells in vitro. The addition of moderate doses of NMDA promoted the survival of wild-type Purkinje cells in the presence of tetrodotoxin. Feeder layers of cerebellar granule cells derived from wild-type or NR1-/- mice promoted survival of Purkinje cells to a similar degree, suggesting that the NMDAR in Purkinje cells, but not in other cells, is directly involved in Purkinje cell viability. The results demonstrate that NMDARs transiently produce membrane current in Purkinje cells and may serve as one of the epigenetic factors that support the survival of Purkinje cells in vitro.

Decreased parvalbumin immunoreactivity in surviving Purkinje cells of patients with spinocerebellar ataxia-1.

The distribution of two calcium-binding proteins, calbindin D28k (CaBP) and parvalbumin (PV), was investigated by immunohistochemistry in the brains of three individuals dying of nonneurologic illness and three patients with spinocerebellar ataxia-1 (SCA-1). SCA-1 has recently been proven to be due to an unstable CAG repeat mutation on chromosome 6. In the cerebellum of control individuals the Purkinje cells showed strong immunoreactivity to CaBP. Other cells were CaBP-negative. Parvalbumin was highly localized to Purkinje, basket, stellate, and Golgi cells. All surviving Purkinje cells in SCA-1 were strongly immunoreactive to CaBP. The number of PV-immunoreactive Purkinje cells was markedly reduced in SCA-1. In addition, there was a significant decrease in the intensity of PV immunostaining within the individual Purkinje cells compared with controls. However, in the hippocampus, temporal cortex, and lateral geniculate scattered PV-positive neurons were seen in SCA-1 patients, similar to those in controls. The present results suggest that the decreased PV-immunoreactivity in the surviving Purkinje cells in SCA-1 may reflect biochemical alterations preceding Purkinje cell degeneration.

Altered IGFBP5 gene expression in the cerebellar external germinal layer of weaver mutant mice.

The IGF system components play important roles in cerebellar development as demonstrated by their specific spatial-temporal expression. IGF-I, type I IGF receptor (IGFR-I), IGFBP2 and IGFBP5 mRNA are localized in distinct cell populations, and all are expressed at the highest levels at the peak of Purkinje cell growth, active synaptogenesis and dendritic formation. To understand IGF-I's action at the cellular level, in situ hybridization was employed to investigate the distribution of IGF system gene transcripts in the cerebellum of weaver mutant mice (wv/wv). Although located ectopically, the surviving Purkinje cells express IGF-I mRNA at the same level in wv/wv as in +/+. No alteration in the cellular distribution or mRNA levels was observed with IGFBP2, or IGFR-I mRNAs. However, the pattern of IGFBP5 expression is altered in the external germinal layer of wv/wv mice. Not only is IGFBP5 expressed by more granule cell precursors of wv/wv cerebellum, but its mRNA level is 2.3 fold that of +/+. The altered IGFBP5 gene expression in granule cell precursors may modulate the interaction of IGF-I with IGFR-I in ways that contribute to their massive death occurring in the development of wv/wv cerebellum.

EMG analysis of harmaline-induced tremor in normal and three strains of mutant mice with Purkinje cell degeneration and the role of the inferior olive

1. The effects of intraperitoneal injections of 10 mg/kg harmaline were tested in normal mice and three strains of cerebellar mutant mice with Purkinje cell degeneration. Ten normal (wild-type) mice (+/+), as well as five lurcher (lc/+), six nervous (nr/nr), and eight Purkinje cell degeneration (pcd/pcd) mutants were implanted with chronic electromyogram (EMG) electrodes in the hamstring and quadriceps muscle groups of the right hindlimb. 2. EMGs were recorded in each of the mice during spontaneous activity before and after intraperitoneal injections of 0.3 ml harmaline (10 mg/kg). Spectral analysis was used to quantify the amplitude and frequency of tremor found in the EMGs after harmaline administration. Normal mice responded to harmaline with strong, continuous 11- to 14-Hz tremor. Mutants from the pcd/pcd strain also reacted with continuous tremor, but of lower amplitude and frequency. In contrast, nr/nr mutants exhibited intermittent paroxysmal tremor lasting for only a few seconds, and lc/+ mutants showed no evidence of tremor whatsoever. 3. In order to detect covert tremor that was possibly not revealed by focal intramuscular EMG recordings, several mutant and normal mice were also tested on a suspended platform to which an accelerometer was attached. The results confirmed the findings from EMG recordings. 4. An incidental observation made during the course of this study was that harmaline tremor disappeared from the normal mouse during swimming and reappeared when the animal was withdrawn from the water. 5. Although Purkinje cells appeared to increase both the depth of modulation and the frequency of tremor, the inhibitory action of the cerebellar cortex does not seem to be essential for the generation of tremor. 6. Parasagittal cerebellar sections of the normal, wild-type mice and the three strains of cerebellar mutant mice of various ages were stained with cresyl violet and examined for Purkinje cell degeneration. Purkinje cell degeneration was found to be complete in the pcd/pcd and lc/+ strains. Although an initial examination of parasagittal sections of the nr/nr strain failed to find any surviving Purkinje cells, further examination of sections cut in the coronal plane revealed small clusters of Purkinje cells in the vermal area of the posterior lobe. 7. The retrograde transport of wheat-germ-agglutinin-conjugated horseradish peroxidase (WGA-HRP) pressure-injected into the cerebellar cortex was used to study the olivocerebellar projections in the wild-type mice and the three strains of cerebellar mutant mice.(ABSTRACT TRUNCATED AT 400 WORDS)

Spatial and temporal pattern of Purkinje cell degeneration in shaker mutant rats with hereditary cerebellar ataxia.

Temporal-spatial patterns of surviving Purkinje cells were studied quantitatively in a rat mutant (shaker) with differential hereditary cerebellar ataxia and Purkinje cell degeneration. Shaker rat mutants are characterized behaviorally as mild if they are ataxic or as strong if they have ataxia and tremor. Purkinje cells degenerate in both mild and strong shaker mutants, but the temporal and spatial patterns of cell death are strikingly different. In mild shaker mutants, Purkinje cell death is temporally restricted, with 31-46% of the Purkinje cells in lobules I-IX dying by 3 months of age. Very few Purkinje cells degenerate after this age. Purkinje cell death is spatially random. In lobules I-IX, every second, third, or fourth Purkinje cell degenerates. Purkinje cells in lobule X do not degenerate. In strong shaker mutants, Purkinje cell degeneration is temporally protracted and spatially restricted. By 3 months of age, most Purkinje cells in lobules I-VIa, -b, and -d have degenerated. Numerous Purkinje cells in the paravermis of lobules VIIb-VIII have also degenerated. Surviving Purkinje cells in the vermis and lateral hemisphere of lobules VIIb-VIII are aligned in parasagittally oriented stripes or transversely oriented bands. Purkinje cells continue to degenerate in localized areas of the posterior lobe such that, by 18 months of age, surviving Purkinje cells are limited primarily to lobules VIc, VIIa, IXd, and X. Quantitative analysis indicates that none of the Purkinje cells in these lobules degenerate.

Effects of nervous mutation on purkinje cell compartments defined by Zebrin II and 9- O-acetylated gangliosides expression

The cerebellum is organized into a series of parasagittally aligned bands which are well delineated in the adult mouse by the largely complementary immunostaining of Purkinje cell groups with the monoclonal antibodies Zebrin II (ZII; antigen: aldolase C) and P-path (antigen: 9- O-acetyl gangliosides). We examined the effect of nervous mutation on compartmental organization using these markers and an antibody to calbindin. In nervous mutant, up to 90% of Purkinje cells die in late postnatal development. The size of the cerebellum is about half that of normal, and caudal lobules appear to decrease in size more than anterior ones. Surviving Purkinje cells corresponded to P-path positive ones that were concentrated in two bilateral bands in the vermis and in medial portions of the hemispheres. Only small numbers of ZII positive cells remained, confirming the report by Wassef et al. with Zebrin I antibody. They were primarily located in caudal lobules IX, X and a portion of lobule IV, paraflocculus and flocculus, and their immunoreactivity was weak compared to that of normal. ZII positive cells are dominant in these caudal lobules, while P-path positive cells dominate in rostral lobules in normal mice, and the similar tendency remains in mutant. Thus, the nervous gene action respects not only sagittal compartments delineated by two antibodies, but also rostro-caudal gradient. The cause of the dominant survival of P-path positive cells awaits future study.

Lurcher purkinje cells express glutamic acid decarboxylase and calbindin mRNAs

Purkinje neurons in immature Lurcher (Lc/+) mice are destined to die as a result of a defect intrinsic to the dying cells. We have used in situ hybridization to determine whether the Lc allele interferes with the normal program of gene expression in the doomed Purkinje cells. In P21 mice, degeneration of Purkinje cells is well underway, but the surviving Purkinje cells continue to express the mRNAs for both glutamate decarboxylase and calbindin D28K, two proteins whose expression is characteristic of normal Purkinje neurons. We conclude that the Lc allele probably does not interfere with the developmental program but acts to cause cell death in already differentiated Purkinje neurons.

Staggerer Mutant Mouse Purkinje Cells Do Not Contain Detectable Calmodulin mRNA

Within the cerebellum calmodulin mRNA is found predominantly in Purkinje cells, with lower levels in granule cells and interneurons. The message shows developmental increases during the first 14 days postnatally. Surviving Purkinje cells of the staggerer (sg/sg) mutant, which are grossly stunted and lack tertiary dendritic spines, contain no detectable calmodulin mRNA, as assayed by Northern blot or an enhanced biotinylated in situ hybridization. This is in contrast to both the Lurcher Purkinje cells and sg/sg granule cells, which express normal levels of this mRNA up until the time they disappear. The sg/sg phenotype can be explained by a defective Purkinje-cell-specific regulatory mechanism for calmodulin.

Chimeric Analysis of the Site of Lurcher Gene Action with Respect to Glial Enzyme Expression

Immunocytochemical analysis of aggregation chimeras made using either of the mutants, Lurcher or Purkinje cell degeneration, previously showed that only Bergmann glia close to surviving Purkinje cells expressed an apparently normal level of the enzyme, glycerol-3-phosphate dehydrogenase (GPDH) (Fisher and Mullen, 1988). In the present study, aggregation chimeras were made using embryos from a B6D2 hybrid mouse strain carrying the Lurcher (Lc/+) mutation and homozygous for an allele specifying a high level of beta-glucuronidase activity and embryos from a wild-type C3H strain with low beta-glucuronidase activity. Chimeric cerebella were analyzed immunocytochemically and histochemically to determine whether Lurcher mutant Bergmann glia could express a normal, high level of GPDH. Comparisons between pairs of alternately stained, 2 microns thick serial sections showed that Bergmann glia with high level of beta-glucuronidase (i.e., Lurcher) expressed normal GPDH immunoreactivity only when close to surviving, wild-type Purkinje cells. Interestingly, in Purkinje cell-free areas of cerebellar cortex that were sufficiently large that GPDH expression was diminished, Bergmann glia also showed a reduced level of histochemically detectable beta-glucuronidase activity, as do Bergmann glia in adult Lc/+ mice. The results indicate that Lc/+ mutant Bergmann glia are not intrinsically defective with regard to expression of the enzymes, GPDH and beta-glucuronidase, but rather, expression of these enzymes depends on glial interaction with wild-type Purkinje cells.

Dendritic and axonal distribution of the microtubule-associated proteins MAP 2 and tau in the cerebellum of the nervous mutant mouse

The fate of the different types of axons and dendrites in the nervous mutant mouse has been studied with antibodies raised against the two major microtubule-associated proteins, MAP 2 and tau. These proteins are specific markers of dendrites and axons, respectively. (1) Immunoblot analysis of cerebellar extracts showed that MAP 2 concentration is markedly reduced (by approximately 90%) in the adult mutant. A 60% decrease was already noticed at day 20 postnatal, i.e., when all the Purkinje cells are present in their normal location and in apparent normal number. (2) Immunohistochemical studies performed at an adult stage with anti MAP 2 antibodies showed marked alterations in the shape of the dendrites of the rare surviving Purkinje cells present in the lateral sections of the cerebellum of the mutant. In the vermis, where 50% of the cells survive in adulthood, the MAP 2 antibody revealed both clusters of cells with a normal density and an intricated and extensive pattern of dendritic arborization and isolated cells showing either an apparently normal or an altered dendritic tree. (3) At day 20 postnatal the same antibody revealed, in the lateral sections severe abnormalities of the dendrites of the Purkinje cells which were different from those seen in adulthood in the vermis. Thus, although few or any Purkinje cells are dead at this stage, a large proportion of them have already profound dendritic alterations. In contrast, in the vermis the Purkinje cells and their dendritic tree are undistinguishable at this stage from those of the unaffected normal mice. (4) Immunoblot and immunohistochemical studies performed with the anti Tau antibody suggested that the majority of the axonal fibers of the cerebellum were present both at day 20 postnatal and at later adult stages. This suggests that, although deprived of their postsynaptic targets these axons can survive for a long time after Purkinje cell death. However, an anti-neurofilament monoclonal antibody which stains specifically the axons of the basket cells, revealed an altered morphology of the basket cell nest in the regions devoid of Purkinje cells. (5) In conclusion the alterations in the morphology of dendrites seem to represent an early event of Purkinje cell degeneration and to be correlated with a marked decrease in expression of MAP 2. It remains unclear, however, whether such changes in expression of MAP 2 represent a primary effect of the mutation or if it is only a precocious result of Purkinje cell degeneration.

Neuronal influence on glial enzyme expression: Evidence from chimeric mouse cerebellum

Cerebella, variably deficient in Purkinje cells, were obtained from aggregation chimeras of either Lurcher or Purkinje cell degeneration mutants. These cerebella were used to analyze the expression of glycerol-3-phosphate dehydrogenase (GPDH) in Bergmann glia. Immunocytochemistry showed apparently normal GPDH expression only in Bergmann glia in the immediate vicinity of surviving Purkinje cells. The number of GPDH-positive Bergmann glia cells associated with isolated Purkinje cells was close to that expected, based on measurements in Golgi-stained, normal cerebella of the Bergmann glia cell's domain. The results support the hypothesis that GPDH expression in Bergmann glia cells depends upon their sustained interaction with Purkinje cells, most likely involving direct cell-cell contact.

Cerebellar mutations affecting the postnatal survival of Purkinje cells in the mouse disclose a longitudinal pattern of differentially sensitive cells

The pattern of surviving Purkinje cells (PCs) was investigated in three cerebellar mutant mice with severe postnatal PC death. Two of these mutations, nervous ( nr) and Purkinje cell degeneration ( pcd) mutations are already well characterized. The third mutation is a new one, which appeared spontaneously in DW/J-Pas mice and was called tambaleante ( tbl). PCs were identified by immunocytochemistry using an antibody against vitamin D-dependent calcium-binding protein which labels all the PCs in adult control mice. In each of the three mutations, surviving PCs are arranged according to a different and reproducible pattern which is symmetric relative to the midline. In NR and young PCD mutants, PCs are closely packed in broad sagittal bands. In TBL, they are more loosely arranged in a rather patchy pattern. In PCD and in TBL mutants the death of resistant PCs is only shortly delayed but in NR there is little change in the number of surviving PCs after 3 months. The differential sensitivity of subsets of PCs to the effect of nr, pcd, and tbl mutations is topographically determined. These results provide a new evidence of the PC heterogeneity which has been previously demonstrated by histochemical and immunohistochemical techniques. Moreover, in the anterior vermis of control mice, three thin sagittal bands of PCs are labeled by the Q113 monoclonal antibody. Similarly, in the anterior lobe of the NR cerebellum, the thin longitudinal strips of missing PCs coincide with the absence of Q113 immunoreactivity: in this region the nr mutation affects specifically the survival of Q113 positive cells. However, other clusters of Q113 immunoreactive PCs do survive in NR mice suggesting that susceptibility to the nr mutation and Q113 positivity are two independent markers of the underlying PC compartmentalization.

Cell lineage dependent and independent control of Purkinje cell number in the mammalian CNS: Further quantitative studies of lurcher chimeric mice

Recent quantitative studies of lurcher chimeric mice have shown that the adult population of cerebellar Purkinje cells can properly be described as a small number of developmental clones of cells. The clones are not seen as patches of contiguous neurons; rather, the cells of any one clone distribute throughout the half-cerebellum that contains them, intermingling extensively with the Purkinje cells of other linkages. Lurcher ⇌ wild-type chimeras were analyzed using the cell autonomous Purkinje-cell-lethal mutant, lurcher ( + L ), as a cell marker. Cell counts from these chimeras revealed that the number of surviving Purkinje cells was always an integral multiple of a unit clone size. These numerical quanta are the evidence for the existence of Purkinje cell developmental clones. When two different inbred strains of mouse were compared (C3H/HeJ and C57BL/6), the resulting clonal analysis showed that the unit clone size (i.e., the number of Purkinje cells in one quantum) is an autonomous property of the lineage and hence, presumably, intrinsic to the progenitor cell that founded it. The current study uses the lurcher chimeric mouse system to examine the cell lineage relationships among the Purkinje cells of a third inbred strain of mouse, AKR/J. The data both support and extend our previous studies. Quantitative analysis reveals that the Purkinje cells of this strain also exist in clones, and the size of these clones is also strain-specific. The number of cells in a single clone (7850), however, is different from either C3H/HeJ (10,200) or C57BL/6 (9200). The fact that this value is so highly polymorphic among the inbred strains of mouse makes it likely that, rather than being a function of different alleles at a single genetic locus, clone size may well represent a multifactorial (but still cell-autonomous) property of developing Purkinje cells. Additional results from a single chimeric animal suggest strongly that clone number (i.e., the number of progenitors selected to found the population) is not strain-specific but results instead from cell:cell interactions during early central nervous system formation.

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

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.

Stereologic study of Purkinje cells in mice after early exposure to phenobarbital

The present investigation was designed to study the dendritic tree of the Purkinje cells surviving after prenatal (Pre B) or neonatal (Neo B) exposure to phenobarbital (PhB). Prenatal exposure in mice was accomplished transplacentally by feeding the pregnant mother 3 g PhB/kg milled food on getation days 9 to 18. Neonatal exposure was conducted directly by injecting the neonates daily (50 mg PhB/kg) on postnatal days 2 to 21. Brains were removed at 14, 21, and 50 days of age. They were cut in the sagittal plane and prepared according to the Golgi method for analysis of the Purkinje cells dendrites. A few brains of 50-day-old Neo B and controls were cut and stained with hemotoxylin and eosin for the assessment of number of Purkinje cells. Neonatal PhB exposure caused 9% reduction in the number of dendritic spines per millimeter at age 14 and 21 days. This deficit was only transient as it disappeared by day 50. However, when the injection dose was reduced to 40 mg PhB/kg the deficits persisted to day 50. Possibly, selection against the most affected individuals accounted for the lesser effect of the higher dose. Prenatal PhB exposure had no long-lasting effect on the dendritic spines. No deficit in the area of the dendritic tree or the number of branches of all orders could be detected in any of the PhB-treated groups studied. Early PhB administration which had severe effects on the neuronal number, had a relatively small effect on the dendrites of the surviving Purkinje cells. Unlike some milder insults, it did not decrease the ratio of granule cells: Purkinje cells, and it is possible that the dendritic effects were dependent on changes in this ratio.

Effects of lidocaine on the electrophysiological properties of subendocardial Purkinje fibers surviving acute myocardial infarction

An electrophysiologic study was conducted in vitro on the surviving subendocardial Purkinje fibers of the canine left ventricle 20 to 24 h after coronary occlusion. The surviving Purkinje cells displayed a low maximum diastolic potential, reduced action potential amplitude, depressed maximal rate of upstroke and a prolonged action potential duration. Spontaneous discharges of high frequency and induced repetitive activity are consistently observed in the infarcted zone but are rarely seen in the normal tissue. Superfusion of lidocaine hydrochloride in concentrations of 1 × 10 −5 to 1 × 10 −4 m slowed or abolished spontaneous and induced firings. The effective refractory period and effective refractory period relative to the total action potential duration were consistently prolonged by lidocaine in the ischemic fibers. Membrane response curves of ischemic fibers were shifted farther to the right than those of normal fibers. Lidocaine exhibited a differential effect on maximul rate of upstroke ( dV dt ) of ischemic Purkinje fibers while causing minor change in the normal fibers. It is suggested that lidocaine exerts its antiarrhythmic effect by selectively depressing the already depressed conducting fibers in the infarcted heart.

EXPERIMENTAL STUDIES ON THE POSTNATAL DEVELOPMENT OF THE BRAIN. III. CEREBELLAR DEVELOPMENT FOLLOWING LOCALIZED ADMINISTRATION OF ENU

Experimental studies on the postnatal development of the train. III. Cere‐bellar development following localized administration of ENUMicroquantities of N‐ethyl‐N‐nitrosourea (ENU) were injected directly into the cerebellum of 5 and 10 day old rats. It was found that ENU had cytotoxic effects not only on the mitotically active cells of the external granular layer, but also on the non‐mitotic differentiating neurons, including Purkinje cells. Regeneration of the external granular layer, in part due to initial immigration of cells from the normal neighboring folia followed by mitosis, was found to be related to and followed by the recovery of the surviving Purkinje cells. These findings suggested that (a) disorientation and disorganization of the Purkinje cells were due to the treatment and not to the degenerative and regenerative activities of the external granular layer, and that (b) regeneration and increase in the external granular layer resulted from induction by the surviving Purkinje cells. Although microscopically the cerebellum appeared to have regenerated, macroscopically ENU had left lasting effects. The folia, where the carcinogen was injected, showed an absence of secondary folia, and the cerebellum as a whole was small in size.