70-Kilodalton Heat Shock Protein Induction in Cerebellar Astrocytes and Cerebellar Granule Cells In Vitro: Comparison with Immunocytochemical Localization After Hyperthermia In Vivo

Induction of the 70-kDa heat shock protein, hsp70, was evaluated in cultured cerebellar astrocytes and granule cell neurons subjected to a hyperthermic stress, using a monoclonal antibody and an oligonucleotide probe that selectively recognize stress-inducible species of hsp70-related proteins and RNAs, respectively. Immunoblots of cultures enriched in either granule cells or astrocytes, and immunocytochemical localization studies in cocultures of these cell types, demonstrated that hsp70 induction was restricted to the astrocyte population. Amino acid incorporation experiments showed little difference in the loss and recovery of overall protein synthesis activity in these two cell types following transient hyperthermic stress. RNA blot hybridizations confirmed the preferential glial induction of hsp70. In vivo immunocytochemical studies in brains of adult rats following hyperthermia were consistent with earlier observations that suggested a primarily glial and vascular localization of the heat shock response in most brain regions, although the intense immunoreactivity in the cerebellar granule cell layer suggests that there is induction of hsp70 in these neurons under in vivo conditions. These results suggest the potential value of such defined cell cultures in identifying mechanisms responsible for differences in the heat shock response of various cell types in vitro, and in revealing factors that may account for the apparent absence of the stress response in cultured cerebellar granule cell neurons.     

Ultrastructure of rat pituitary LH gonadotrophs in relation to serum and pituitary LH levels following repeated LH-RH stimulation

The effects of single and repeated LH-RH injections at 120 min intervals on female rat LH gonadotrophs and on pituitary and serum LH levels were investigated using electronmicroscopy and radioimmunoassay. A temporary stimulation of granule release, of protein and new granule synthesis and of the accumulation of lysosomal structures was found in LH cells after the first LH-RH injection. The temporary stimulations were massively enhanced after the second injection. These consecutive yet in their time-sequence overlapping processes account for the initial depletion of secretory granule content (3--15 min after LH-RH injection), for the subsequent regranulation and accumulation of granules above control levels (60--120 min after injection) and also for the reduction in the number of granules to control levels (150 min after LH-RH injection and thereafter). Increased polymorphic lysosomal structures are believed to be responsible for this reduction of excess granules. The amount of assayable pituitary and serum LH generally corresponds with the morphological changes observed in LH-gonadotrophs, thus further substantiating the above observations. A schema which summarizes the observed morphological and hormonal changes in their time-sequence in response to LH-RH stimulation depicts the short-term regulation of secretory processes in female gonadotrophs.     

Differential Cellular Enrichment of Gangliosides in the Mouse Cerebellum: Analysis Using Neurological Mutants

Abstract: The cellular distribution of gangliosides in the cerebellum was studied in a series of adult mouse mutants that lose specific populations of neurons. The weaver ( wv ) mutation destroys the vast majority of granule cells, whereas the Purkinje cell degeneration mutation ( pcd ) destroys the vast majority of Purkinje cells. The staggerer ( sg ) and lurcher ( Lc ) mutations, on the other hand, destroy the vast majority of both granule and Purkinje cells. A proliferation of reactive glial cells, which occurs as a consequence of neuronal loss, has been reported in the sg/sg and pcd/pcd mutants, but not in the wv/wv mutant. Compared with the normal (+/+) mice, the concentration (μg/100 mg dry weight) of G_D1a was significantly reduced in those mutants that lost granule cells, but was not reduced in the pcd/pcd mutant. The concentration of G_TIa, on the other hand, was significantly reduced in those mutants that lost Purkinje cells, but was not reduced in the wv/wv mutant. A significant elevation in the concentration of G_D3, which may be related to the proliferation of reactive glial cells, was observed in the pcd/pcd, sglsg , and Lc /+ mutants, but was not observed in the wv/wv mutant. Because these ganglioside abnormalities were confined to the cerebellum, they cannot result from genetic defects in ganglioside metabolism. Instead, these abnormalities result from a differential enrichment of gangliosides in neural membranes. Our findings suggest that G_DT1a is more heavily concentrated in granule cells than Purkinje cells, whereas the opposite appears true for G_Tla. It also appears that GD3 is enriched in reactive glial cells and may play an important role during the morphological transformation of neural membranes.     

Molecular mechanisms of An-Chuan Granule for the treatment of asthma based on a network pharmacology approach and experimental validation

An-Chuan Granule (ACG), a traditional Chinese medicine (TCM) formula, is an effective treatment for asthma but its pharmacological mechanism remains poorly understood. In the present study, network pharmacology was applied to explore the potential mechanism of ACG in the treatment of asthma. The tumor necrosis factor (TNF), Toll-like receptor (TLR), and Th17 cell differentiation-related, nucleotide-binding oligomerization domain (NOD)-like receptor, and NF-kappaB pathways were identified as the most significant signaling pathways involved in the therapeutic effect of ACG on asthma. A mouse asthma model was established using ovalbumin (OVA) to verify the effect of ACG and the underlying mechanism. The results showed that ACG treatment not only attenuated the clinical symptoms, but also reduced inflammatory cell infiltration, mucus secretion and MUC5AC production in lung tissue of asthmatic mice. In addition, ACG treatment notably decreased the inflammatory cell numbers in bronchoalveolar lavage fluid (BALF) and the levels of pro-inflammatory cytokines (including IL-6, IL-17, IL-23, TNF-alpha, IL-1beta and TGF-beta) in lung tissue of asthmatic mice. In addition, ACG treatment remarkably down-regulated the expression of TLR4, p-P65, NLRP3, Caspase-1 and adenosquamous carcinoma (ASC) in lung tissue. Further, ACG treatment decreased the expression of receptor-related orphan receptor (RORγt) in lung tissue but increased that of Forkhead box (Foxp3). In conclusion, the above results demonstrate that ACG alleviates the severity of asthma in a ´multi-compound and multi-target’ manner, which provides a basis for better understanding of the application of ACG in the treatment of asthma.     

Oligodendrocyte ablation affects the coordinated interaction between granule and Purkinje neurons during cerebellum development

Oligodendrocytes (OLs) are the glial cells of the central nervous system (CNS) classically known to be devoted to the formation of myelin sheaths around most axons of the vertebrate brain. We have addressed the role of these cells during cerebellar development, by ablating OLs in vivo. Previous analyses had indicated that OL ablation during the first six postnatal days results into a striking cerebellar phenotype, whose major features are a strong reduction of granule neurons and aberrant Purkinje cells development. These two cell types are highly interconnected during cerebellar development through the production of molecules that help their proliferation, differentiation and maintenance. In this article, we present data showing that OL ablation has major effects on the physiology of Purkinje (PC) and granule cells (GC). In particular, OL ablation results into a reduction of sonic hedgehog (Shh), Brain Derived Neurotrophic Factor (BDNF), and Reelin (Rln) expression. These results indicate that absence of OLs profoundly alters the normal cerebellar developmental program.     

Postnatal Development of a Granule Cell-Enriched, Neurone-Specific Glycoprotein, gp50, in Normal and Thyroid-Deficient Rats

Glycoprotein gp50 is a neurone-specific, granule cell-enriched glycoprotein that is also a major component of isolated synaptic membranes. Here, we describe the use of a monoclonal antibody, mab SM gp50, to study the postnatal development of gp50 in the brain of normal and thyroid-deficient rats. Radioimmunoassay, enzyme-linked immunosorbent assay, and Western blotting show that gp50 is not detectable in brain until postnatal day 4 (P4) in both forebrain and cerebellum. In forebrain, the rate of increase of gp50 levels is maximal between P12 and P20. It is somewhat later in cerebellum, where peak levels are attained between P30 and P35. Immunocytochemical studies show little detectable gp50-like immunoreactivity before P16, and the staining is still weak, relative to adult tissue, at P25. The intense staining of the granule cell layer characteristic of adult cerebellum predominantly appears after P25. Development of gp50 is severely retarded in the cerebellum of thyroid-deficient rats, particularly during the second and third postnatal weeks. However, by the fourth postnatal week, gp50 levels in normal and hypothyroid animals are comparable. The results indicate that significant alterations in the pattern of gp50 expression continue to occur at a late stage of cerebellar development. In particular, the increase in immunocytochemical staining of the granule cells after P25 is striking in that by this time most major events associated with cerebellar development are essentially complete.     

Uptake of Magnesium by Chromaffin Granules In Vitro: Role of the Proton Electrochemical Gradient

Abstract: The chromaffin granule membrane in vitro is impermeable to protons as well as to Mg²⁺; however, when granules are incubated in the presence of the proton ionophore carbonyl cyanide p -trifluoromethoxy-phenylhydrazone or an inhibitor of the granule membrane Mg²⁺-dependent ATPase, the metal ion is accumulated inside the granules. This accumulation is dependent upon the granule transmembrane potential. The simultaneous presence of the ATPase inhibitor and the proton ionophore markedly increases metal ion incorporation. Mg²⁺ incorporation is also promoted by nigericin in the presence of potassium or sodium ions, indicating that Mg²⁺ accumulation is also dependent upon the transmembrane pH gradient. Concomitant with the Mg²⁺ accumulation, there is a significant loss of endogenous catecholamines. It is concluded that Mg²⁺ accumulation is determined by the electrochemical gradient maintained across the membrane. Once the metal ion has accumulated into the granules it displaces catecholamines from their storage sites.     

Developmental changes in the expression pattern of the JNK activator kinase MUK/DLK/ZPK and active JNK in the mouse cerebellum

JNK is one of the key molecules regulating cell differentiation and migration in a variety of cell types, including cerebral cortical neurons. MUK/DLK/ZPK belongs to the MAP kinase-kinase-kinase class of protein kinases for the JNK pathway and is expressed predominantly in neural tissue. We have determined the expression pattern of MUK/DLK/ZPK and active JNK in the cerebellum at different stages of postnatal development. Quantitative analysis by Western blotting has showed that high expression levels of MUK/DLK/ZPK and active JNK are maintained during the postnatal development of the cerebellum, and that these levels decrease in the adult cerebellum. Immunohistochemical staining has revealed, however, that their distribution in the developing cerebellum is considerably different. Although active JNK is highly concentrated in the premigratory zone of the external germinal layer (EGL), high expression of MUK/DLK/ZPK has been observed in the molecular layer and in the premigratory zone. Neither the active JNK nor MUK protein has been detected in the proliferative zone of the EGL. These observations suggest that during the postnatal development of the cerebellum, the MUK-JNK signaling pathway contributes to the regulation of granule cell differentiation and migration; further, the activity of MUK/DLK/ZPK is tightly regulated by posttranslational mechanisms and by its expression level.This work was supported by a Ishizu Shun memorial scholarship and grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan.     

Calbindin D-28k Immunoreactivity and Its Protein Level in Hippocampal Subregions During Normal Aging in Gerbils

The hippocampus is associated with learning and memory function and shows neurochemical changes in aging processes. Calbindin D-28k (CB) binds calcium ion with a fast association rate. We examined age-related changes in CB immunoreactivity and its protein level in the gerbil hippocampus during normal aging. In the hippocampal CA1 region (CA1) and CA2, CB immunoreaction was found in some neurons in the stratum pyramidale (SP) at postnatal month 1 (PM 1). CB immunoreactivity in neurons was markedly increased at PM 3. Thereafter, CB immunoreactivity was decreased with time: CB-immunoreactive (⁺) neurons were fewest at PM 24. In the CA3, a few CB⁺ neurons were found only in the SP at PM 1 and in the stratum radiatum at PM 18 and 24. In addition, mossy fibers were stained with CB at PM 1. CB immunoreactivity in mossy fibers was markedly increased at PM 3, thereafter it was decreased with time. In the dentate gyrus, many granule cells (GC) in the granule cell layer were stained with CB at PM 1. CB immunoreactivity in GC was markedly increased at PM 3, thereafter CB immunoreactivity was decreased with time. In Western blot analysis, CB protein level in the gerbil hippocampus was highest at PM 3, thereafter CB protein levels were decreased with time. This result indicates that CB in the gerbil hippocampus is abundant at PM 3 and is decreased with age.     

Cellular and molecular control of dendritic growth and development of cerebellar Purkinje cells

Purkinje cells are the principal neurons of the cerebellar cortex and are characterized by a large and highly branched dendritic tree. For this reason, they have for a long time been an attractive model system to study the regulation of dendritic growth and differentiation. In this article, I will first review studies on different aspects of Purkinje cell dendritic development and then go on to present studies which have aimed at experimentally altering Purkinje cell dendritic development. Some of the cellular and molecular mechanisms which have been shown by these studies to be important determinants of Purkinje cell dendritic development will be discussed, in particular the role of the parallel fiber input, of hormones, and of neuronal growth factors. The organotypic slice culture method will be introduced as an important experimental tool to study Purkinje cell dendritic development under controlled conditions. Using cerebellar slice cultures, protein kinase C (PKC) has been identified as a major determinant of Purkinje cell dendritic development and the contribution of specific isoforms of PKC will be discussed. Finally, it will be shown that Purkinje cell dendritic development in slice cultures does not depend on the activation of glutamate receptors and appears to be independent of the presence of the neurotrophin BDNF. These studies indicate that the initial outgrowth of the Purkinje cell dendritic tree can occur in the absence of signals derived from afferent fibers, but is under control of PKC signaling.