The insulin-like growth factor I system in the rat cerebellum: Developmental regulation and role in neuronal survival and differentiation

The developmental regulation of insulin-like growth factor I (IGF-I), its receptor, and its binding proteins (IGFBPs) was studied in the rat cerebellum. All the components of the IGF-I system were detectable in the cerebellum at least by embryonic day 19. Levels of IGF-I receptor and its mRNA were highest at perinatal ages and steadily decrease thereafter, although a partial recovery in IGF-I receptor mRNA was found in adults. Levels of IGF-I and its mRNA also peaked at early ages, although immunoreactive IGF-I showed a second peak during adulthood. Finally, levels of IGFBPs were also highest at early postnatal ages and abruptly decreased thereafter to reach lower adult levels. Since highest levels of the different components of the IGF-I system were found at periods of active cellular growth and differentiation we also examined possible trophic effects of IGF-I on developing cerebellar cells in vitro. We found a dose-dependent effect of IGF-I on neuron survival together with a specific increase of the two main neurotransmitters used by cerebellar neurons, GABA and glutamate. Analysis of cerebellar cultures by combined in vitro autoradiography and immunocytochemistry with cell-specific markers indicated that both Purkinje cells (calbindin-positive) and other neurons (neurofilament-positive) contain IGF-I binding sites. These results extend previous observations on a developmental regulation of the IGF-I system in the cerebellum and reinforce the notion of a physiologically relevant trophic role of IGF-I in cerebellar development. © 1994 Wiley-Liss, Inc.     

Somatostatin gene expression in the developing monkey frontal and cerebellar cortices

Somatostatin (SRIF) mRNA was determined in the developing monkey frontal and cerebellar cortices by the dot blot and the northern blot analyses at embryonic day 120 (E120), embryonic day 140 (E140), newborn stage (Nb), postnatal day 60 (P60) and adult stage (Ad.) At E120, at which time the migration of the cortical neurons had already been completed, SRIF mRNA was detectable with 50% of the maximal value at E140 in the cerebral frontal cortex (von Bonin and Bailey's area FD). After E140, the level of mRNA gradually declined to the adult level by P60 with 25% of the maximal value. In the cerebellum, SRIF mRNA was highly expressed at E120. The level decreased to 18% of the maximum at E140. Between the newborn and adult stages, there existed no positive signal of the mRNA. In contrast, both fetal and adult liver tissues contained no amounts of SRIF mRNA. We discussed the physiological meanings of the enhanced SRIF gene expression in the developing monkey cerebral and cerebellar cortices.     

Receptor-mediated regulation of neuropeptide gene expression in astrocytes

One of the functions of glial receptors is to regulate synthesis and release of a variety of neuropeptides and growth factor peptides, which in turn act on neurons or other glia. Because of the potential importance of these interactions in injured brain, we have examined the role of two different receptors in the regulation of astrocyte neuropeptide synthesis. Stimulation of β-adrenergic receptors on type 1 astrocytes resulted in increased mRNA and protein for the proenkephalin (PE) and somatostatin genes. This receptor also increased expression of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). The potential role of opiate receptors was examined in several ways. Treatment of newborn rats for 7 days with the opiate antagonist naltrex-one, prior to preparation of astrocytes, had no effect on PE mRNA or met-enkephalin content but resulted in a significant increase in NGF content. However, treatment of astrocytes in culture with met-enkephalin, morphine, or naltrexone had no effect on any of these parameters. No opiate binding could be detected, using either etorphine or bremazocine, to membranes of astrocytes prepared from cortex, cerebellum, striatum, or hippocampus of 1-day, 7-day, or 14-day postnatal rats. Thus we conclude that type 1 astrocytes do not express opiate receptors and that the in vivo effects of naltrexone are mediated indirectly via some other cell type/receptor. © 1994 Wiley-Liss, Inc.     

Expression of DA11, a neuronal-injury-induced fatty acid binding protein,coincides with axon growth and neuronal differentiation during central nervous system development

DA11 is the first fatty acid binding protein (FABP) for which gene expression has been shown to be upregulated following neuronal injury in the adult peripheral nervous system. To understand better the potential regulatory role(s) of this unique FABP in axonal growth and neuronal differentiation, we undertook a temporal and spatial study of DA11 gene expression in the developing rat central nervous system (CNS). Transient upregulation of DA11 mRNA and protein levels in CNS tissues were quantified by Northern blot hybridization and Western immunoblot analyses at different developmental ages. Homogenates of embryonic and neonatal cerebral cortex, cerebellum, brainstem, and hippocampal tissues contained 100-fold more DA11 mRNA and protein than corresponding adult tissues. Significant increase in DA11 mRNA was observed as early as embryonic day (E) 14 in cerebral cortex and cerebellum and E19 in brain stem and hippocampus. Postnatal levels of DA11 remained elevated through postnatal day (P) 10 in cerebral cortex, P14 in brain stem and hippocampus, and P20 in cerebellum. Localization of DA11-like immunoreactivity to specific CNS tissues, cell types, and intracellular compartments at P9 revealed a spatial pattern of neuronal expression different than that reported for other FABPs. DA11 protein was detected in the nucleus, cytoplasm, axons, and dendrites of differentiating neurons in cerebral cortex, hippocampus, cerebellum, brain stem, spinal cord, and olfactory bulb. The strong association of DA11 gene expression with development throughout the CNS suggests that this unique FABP plays an important role in axonal growth and neuronal differentiation in many different neuronal populations. J. Neurosci. Res. 48:551–562, 1997. © 1997 Wiley-Liss Inc.     

Calcium requirements for barium stimulation of enkephalin and vasoactive intestinal peptide biosynthesis in adrenomedullary chromaffin cells

The divalent cation barium was used to study the role of calcium in coupling neuropeptide secretion and biosynthesis following secretagogue stimulation of bovine chromaffin cells. Barium chloride (0.1-2.5 mM) stimulated in a dose-dependent manner the secretion of met-enkephalin (up to 20% of intracellular peptide content) and increased the total amount (cell plus medium content) of met-enkephalin and vasoactive intestinal polypeptide (VIP) 2- to 3-fold after 72 hours. A greater than six-fold increase in proenkephalin mRNA (mRNA(enk)) was observed by 24 hours following barium stimulation. The voltage-sensitive calcium channel blocker D600 inhibited the barium-stimulated secretion of enkephalin and blocked the stimulation of VIP biosynthesis and mRNA(enk). Reducing calcium in the medium resulted in an enhancement of barium-stimulated release of both peptides, but blocked the induction of their biosynthesis. The data indicate that calcium targets involved in secretion can be activated by barium or calcium while calcium targets involved in biosynthesis specifically require calcium. It is therefore proposed that pathways leading to peptide secretion and biosynthesis in the adrenal diverge just after secretagogue-stimulated calcium influx.     

Neurons exposed to ammonia reproduce the differential alteration in nitric oxide modulation of guanylate cyclase in the cerebellum and cortex of patients with liver cirrhosis

The activation of soluble guanylate cyclase by nitric oxide is increased in the frontal cortex but is reduced in the cerebellum of patients who died with liver cirrhosis. The aims of this work were to assess whether hyperammonemia is responsible for the region-selective alterations in guanylate cyclase modulation in liver cirrhosis and to assess whether the alteration occurs in neurons or in astrocytes. The activation of guanylate cyclase by nitric oxide was lower in cerebellar neurons exposed to ammonia (1.5-fold) than in control neurons (3.3-fold). The activation of guanylate cyclase by nitric oxide was higher in cortical neurons exposed to ammonia (8.7-fold) than in control neurons (5.5-fold). The activation was not affected in cerebellar or cortical astrocytes. These findings indicate that hyperammonemia is responsible for the differential alterations in the modulation of soluble guanylate cyclase in cerebellum and cerebral cortex of cirrhotic patients. Moreover, the alterations occur specifically in neurons and not in astrocytes.     

Gene expression of OGFr in the developing and adult rat brain and cerebellum

The native opioid peptide, [Met5]-enkephalin (termed opioid growth factor (OGF)), is a tonically active negative growth factor targeted to cell proliferation in the developing nervous system. OGF action is mediated by the OGF receptor (OGFr). The present study investigates gene expression of OGFr in the developing and adult brain and cerebellum of the rat using Northern blot analysis and normalization to GAPDH. OGFr was detected in whole brain at embryonic day 20 and birth, and was at least twofold greater than neonatal levels during the first week of life. From postnatal day 15 onwards to adulthood, levels of OGFr mRNA in the whole brain were detectable but less than those at birth. OGFr mRNA in cerebellum was found on embryonic day 20, and remained relatively constant until postnatal day 12 when a sharp increase was recorded. In the third week of life and continuing into adulthood, cerebellar OGFr mRNA was detected at levels comparable to those in postnatal week 1. These results show that message for OGFr is developmentally regulated prior to and after birth, is ubiquitously expressed during development, and is present in the adult brain and cerebellum even though OGF receptor binding is not recorded.     

Alterations in somatostatin and proenkephalin mRNA in response to a single amygdaloid stimulation versus kindling.

Previous studies have shown changes in both somatostatin (SS)- and proenkephalin(PE)-derived peptides in the brains of amygdaloid-kindled rats, suggesting possible roles for the peptides in the kindling process. In this study, we have extended this analysis by looking at the time course of changes in SS and PE mRNAs at various times after kindling, in comparison with a single non-convulsive stimulation. Blot analysis of total RNA showed increases in SS mRNA in striatum, frontal cortex and hippocampus of animals receiving only a single stimulation as well as kindled animals--the increase occurred 1-3 days following stimulation and levels were back to basal by 1 week. PE mRNA did not change. In situ hybridization analysis, one day after the last kindling stimulation, showed significant elevations of SS mRNA in CA1, CA2 and dentate gyrus of hippocampus and of PE mRNA in olfactory cortex that were specific to kindling. However, both a single stimulation and kindling increased PE mRNA in olfactory tubercle and arcuate nucleus. In contrast, a single electrical stimulus increased PE mRNA in ventral striatum and SS mRNA in cingulate cortex and olfactory tubercle. These data support the idea that changes of SS mRNA in hippocampus and of PE mRNA in olfactory cortex may be related to kindling, and point out the importance of using animals which receive a single electrical stimulus, rather than sham-operated animals, as controls.     

Preprotachykinin A mRNA expression in the rat brain during development

Expression of preprotachykinin A (PPT-A) mRNA was analyzed by northern blots using mRNA prepared from rat brain at 12 different developmental stages ranging from embryonic day 15 (E15) to adult. A single PPT-A mRNA of 1.3 kb was detected throughout development. PPT-A mRNA was detected as early as E15 and an approximately 3-fold increase occurred at birth. This amount remained until 3 weeks of age when the level increased, reaching a peak at 5 weeks of age. Adult amounts were approximately 3-fold higher than the levels at birth. The distribution of PPT-A mRNA-expressing cells in rat brain was studied by in situ hybridization on sections from embryonic day 20, postnatal days 4 and 7 as well as adult. Cells expressing PPT-A mRNA were detected in the forebrain at all 4 ages analyzed. However, the hybridization pattern and the labeling intensity varied in different brain regions during development. In cingulate cortex, intense labeling was seen in numerous cells at embryonic day 20 and postnatal days 4 and 7, whereas in the adult cingulate cortex only a few scattered labeled cells were observed. In frontoparietal cortex labeled cells were found from postnatal day 4 to adult, with the highest density of labeled cells at P7. Developmental differences in both the distribution of PPT-A mRNA-expressing cells and the level of PPT-A mRNA expression were also found in caudate-putamen, lateral hypothalamus and amygdala. Thus, our results show several changes in PPT-A mRNA expression during ontogeny, indicating a region and time-specific regulation of PPT-A mRNA expression during brain maturation.     

Embryonic cerebellar astroglia in vitro

Three types of astroglia appear during cerebellar development--radial glia and Bergmann glia, which are thought to facilitate neuronal migration, and astrocytes, which are thought to compartmentalize mature granule neurons. Cells resembling Bergmann glia and astrocytes have been described in cultures of cerebellar cells harvested from early postnatal cerebellum. In this study, we have used cell-type specific antisera to visualize embryonic forms of cerebellar astroglia and their interaction with embryonic neurons in vitro. When cells were dissociated from mouse cerebellum on the thirteenth embryonic day (E13), 3 forms of cells were stained with antisera raised against purified glial filament protein ( AbGF ), all of which had more elongated processes and less complex shapes than astroglia from postnatal day 7. The vast majority of embryonic cerebellar neurons did not contact these immature forms of astroglia.     

Tissue plasminogen activator mRNA expression in granule neurons coincides with their migration in the developing cerebellum

Tissue plasminogen activator activity in the developing cerebellum, as quantified by zymography of cerebellar homogenates from embryonic day (E) 17 to adult mice, shows a peak of activity at postnatal day (P) 7, followed by a steady 75% decrease into adulthood. Northern blot analysis reveals a similar pattern for tissue plasminogen activator mRNA levels, which are low at E17 but increase dramatically, reaching their highest levels of specific mRNA/μg RNA in P1–P7 mice and declining about threefold in the adult mouse. In situ hybridization of whole mouse brain sections with a tissue plasminogen activator antisense cRNA probe shows pronounced reactivity in the cerebellum. Although some binding is associated with the cerebellar meninges, the external granule layer is devoid of tissue plasminogen activator mRNA at all ages. However, highly labeled elongated cells, which also bind antibody to neuronal nuclear antigen and are adjacent to Bergmann glial fibers (i.e., migrating granule neurons), are readily visible throughout the molecular and Purkinje layers at P7 and P14. In the adult mouse cerebellum, tissue plasminogen activator mRNA labeling is restricted to cells in the Purkinje/internal granule layers. Thus, tissue plasminogen activator gene expression is induced as granule neurons leave the external granule layer and begin their inward migration. © 1995 Wiley-Liss, Inc.     

Corticotropin releasing factor induces proliferation of cerebellar astrocytes

In the adult cerebellum, corticotropin releasing factor (CRF), that is localized in climbing fibers, mossy fibers, and a fine varicose plexus along the Purkinje cell layer, modulates the responsiveness of Purkinje cells to excitatory amino acids. During development, CRF has been detected in the primitive cerebellar anlage as early as embryonic day (E)10, and is continuously expressed throughout embryonic and postnatal cerebellar ontogeny. To investigate a possible trophic role for CRF during cerebellar development, cerebellar culture studies using E18 mouse embryos were carried out. In our culture paradigm, that used serum-free defined medium to suppress cell proliferation, CRF induced proliferation of cells in a dose-dependent manner in a range of concentrations between 0.1-10 microM. The proliferating cells were identified as astrocytes based on their expression of vimentin and GFAP. BrdU incorporation studies supported the proposed mitogenic effect of CRF on developing astrocytes. The mitogenic effects of CRF seemed to be primarily on immature astrocytes determined by their differential expression of vimentin and GFAP. Astrocytes at more advanced stages of development, as determined by the extent of process outgrowth and GFAP expression, incorporated less BrdU compared to immature astrocytes. CRF receptors were localized in astrocytes, and the proliferation of astrocytes induced by CRF was inhibited by astressin, a competitive CRF receptor antagonist. In conclusion, CRF induces proliferation of astrocytes derived from the developing cerebellum, that suggests a gliotrophic role for CRF during cerebellar development.     

Close correlation between the birth date of Purkinje cells and the longitudinal compartmentalization of the mouse adult cerebellum

The adult cerebellum is organized into longitudinal compartments that are revealed by specific axonal projections (olivocerebellar and corticonuclear projections). These compartments in the adult cerebellum are closely correlated with the striped expression of zebrin II (aldolase C), a late-onset marker of Purkinje cells. Similarly, the embryonic cerebellum is organized into longitudinal compartments that are revealed by striped expression of other genes (early-onset markers). The cerebellar compartments are thought to be the basic and functional subdivisions of the cerebellum. However, the relationship between the embryonic (early-onset) and the adult (late-onset) compartments has remained unknown, because the pattern of the embryonic compartments is distinct from that of the adult compartments. To examine this issue, we labeled Purkinje cells (PCs) born at embryonic day (E) 10.5, E11.5, and E12.5 by using an adenoviral vector and traced their fated positions in the adult cerebellum. By comparing the striped distribution of each cohort of birth date-related PCs with the striped pattern of zebrin II immunoreactivity (zebrin II bands) in the entire adult cerebellum, we found that the striped distribution of PCs correlated strikingly with zebrin II bands. Generally, a single early-onset compartment was transformed directly into a single late-onset compartment. Therefore, our observation also indicated the close correlation between the compartments formed by birth date-related PCs and olivocerebellar projections. Furthermore, we found that the cerebellum was composed of three units showing lateral-to-medial developmental gradients, as revealed by the birth dates of PCs. The results suggest that PC birth dates play an important role in organizing cerebellar compartmentalization.     

Developmental expression of proenkephalin mRNA in rat striatum and in striatal cultures

Proenkephalin mRNA shows a biphasic developmental profile in rat striatum, with an initial peak at postnatal day 2, a decline to embryonic levels by day 7, and a second increase to adult levels over the course of the second to 4th week after birth. The same 4-fold increase is seen in cultured striatal neurons, over the same time course but without a biphasic response. Cultured fetal glia also contain proenkephalin mRNA.     

Development of the cerebellar cortical efferent projection: an in-vitro anterograde tracing study in rat brain slices.

This study reports the results of an anterograde HRP analysis of the development of cerebellar cortical efferents studied in-vitro with cerebellum-brainstem slices from embryonic (E-18) through newborn rats. From E-18, Purkinje cells in the medial region of the cerebellum are seen to project to the medial cerebellar and vestibular nuclei, their appropriate targets in the adult animal. Thus, the cortical efferent system is forming during late embryogenesis and may establish synaptic contacts with target cells as early as E-20.     

Developmental appearance of somatostatin in the rat cerebellum: in situ hybridization and immunohistochemistry

The postnatal appearance of the mRNA for preprosomatostatin, as well as the presence of somatostatin 28 and somatostatin 28, was examined in the rat cerebellum. RNA blotting analysis indicates a dramatic decrease in the level of this mRNA during cerebellar maturation. In situ hybridization reveals that many cells contain this mRNA at early postnatal stages, with progressively fewer cells being labeled after postnatal day 20. Immunohistochemical analysis indicates that there is a changing pattern in the distribution of these somatostatin peptides through cerebellar development. Initially, cells in the granule cell layer are stained, and fiber immunoreactivity is localized primarily in this same layer. During the second postnatal week, this granule cell layer staining decreases, and some Purkinje cells display immunoreactivity, particularly in the flocculus and paraflocculus. This Purkinje cell staining decreases by postnatal day 30, but immunoreactive cells are still present in the granule cell layer. In contrast, in the adult cerebellum, very few of these cells remain immunoreactive.     

Heterogeneous expression of carboxypeptidase E and proenkephalin mRNAs by cultured astrocytes.

Cultured astrocytes have been found to express neuropeptides, neuropeptide processing enzymes and their mRNAs. Although astrocytes were shown to display regional variation in their expression of these mRNAs, it was unclear whether all astrocytes cultured from the same brain region express similar mRNA levels or if this expression is heterogeneous. We examined the individual heterogeneity of astrocytes cultured from several brain regions by in situ hybridization. Astrocytes derived from the frontal cortex, hypothalamus and cerebellum of neonatal rat brains were cultured for 3 weeks and then analyzed by in situ hybridization using 35S-labeled cRNA probes to carboxypeptidase E (CPE), proenkephalin (PE), and cyclophilin (1B15) mRNAs and an oligomeric probe complimentary to the first 45 bases of rat 18S rRNA. Frequency histograms generated by counting the grains produced over emulsion-coated cells demonstrated populations of astrocytes expressing high levels of mRNA for CPE and PE and a population expressing low to background levels of these mRNAs. In contrast, all cultured astrocytes expressed high levels of 1B15 mRNA and 18S rRNA. The percentage of cultured astrocytes expressing high levels of CPE mRNA was 42% for frontal cortex astrocytes and 23% for cerebellar astrocytes. While the percentages of cultured astrocytes expressing high levels of PE mRNA varied slightly between brain region (24-28%), the level of PE mRNA expression per cell showed greater variation between regions. The effect of culture density on the expression of PE mRNA was also examined. Approximately 55% of the cells in low density cultures expressed PE mRNA, while only 5-10% of the cells in high density cultures expressed this mRNA. These results indicate that cultured astrocytes display individual heterogeneity with regard to neuropeptide biosynthesis and that the expression of neuropeptides by these cells is regulated.