Maturation of astrocyte morphology and the establishment of astrocyte domains during postnatal hippocampal development

Mature protoplasmic astrocytes exhibit an extremely dense ramification of fine processes, yielding a 'spongiform' morphology. This complex morphology enables protoplasmic astrocytes to maintain intimate relationships with many elements of the brain parenchyma, most notably synapses. Recently, it has been demonstrated that astrocytes establish individual cellular-level domains within the neuropil, with limited overlap occurring between the extents of neighboring astrocytes. The highly ramified nature of protoplasmic astrocytes is closely associated with their ability to create such domains. This study was an attempt to characterize the development of spongiform processes and the establishment of astrocyte domains. A combination of immunolabeling for the astrocyte-specific markers glial fibrillary acidic protein and S100beta with intracellular dye labeling in fixed tissue slices allowed for the identification of immature astrocytes and the elucidation of their complete, well-preserved morphologies. We find that during the first two postnatal weeks astrocytes extend stringy, filopodial processes. Fine, spongiform processes appear during the third week. Protoplasmic astrocytes are quite heterogeneous in morphology at 1-week postnatum, but there is a remarkable consistency in morphology by 2 weeks of age. Finally, protoplasmic astrocytes initially extend long, overlapping processes during the first two postnatal weeks. The subsequent elaboration of spongiform processes results in the development of boundaries between neighboring astrocyte domains. Stray processes that encroach on neighboring domains are eventually pruned by 1 month of age. These observations suggest that domain formation is largely the consequence of competition between astrocyte processes, similar to the well-studied competitive interactions between certain neuronal dendritic fields.     

Hypothyroidism affects astrocyte and microglial morphology in type 2 diabetes

In the present study,we investigated the effects of hypothyroidism on the morphology of astrocytes and microglia in the hippocampus of Zucker diabetic fatty rats and Zucker lean control rats.To induce hypothyroidism,Zucker lean control and Zucker diabetic fatty rats at 7 weeks of age orally received the vehicle or methimazole,an anti-thyroid drug,treatment for 5 weeks and were sacrificed at 12 weeks of age in all groups for blood chemistry and immunohistochemical staining.In the methimazole-treated Zucker lean control and Zucker diabetic fatty rats,the serum circulating triiodothyronine(T3)and thyroxine(T4)levels were significantly decreased compared to levels observed in the vehicle-treated Zucker lean control or Zucker diabetic fatty rats.This reduction was more prominent in the methimazole-treated Zucker diabetic fatty group.Glial fibrillary acidic protein immunoreactive astrocytes and ionized calcium-binding adapter molecule 1(Iba-1)-immunoreactive microglia in the Zucker lean control and Zucker diabetic fatty group were diffusely detected in the hippocampal CA1 region and dentate gyrus.There were no significant differences in the glial fibrillary acidic protein and Iba-1 immunoreactivity in the CA1 region and dentate gyrus between Zucker lean control and Zucker diabetic fatty groups.However,in the methimazole-treated Zucker lean control and Zucker diabetic fatty groups,the processes of glial fibrillary acidic protein immunoreactive astrocytes and Iba-1 immunoreactive microglia,were significantly decreased in both the CA1region and dentate gyrus compared to that in the vehicle-treated Zucker lean control and Zucker diabetic fatty groups.These results suggest that diabetes has no effect on the morphology of astrocytes and microglia and that hypothyroidism during the onset of diabetes prominently reduces the processes of astrocytes and microglia.     

A critical role for palladin in astrocyte morphology and response to injury

Astrocytes respond to injury of the CNS with a dramatic change in morphology, contributing to the formation of a glial scar. We recently identified a novel actin-associated protein named palladin, which possesses the features of a potent cytoskeletal scaffold. Palladin expression was assayed in two populations of cultured astrocytes, polygonal versus stellate, and was detected at high levels in polygonal astrocytes and low levels in stellate astrocytes. When stellate astrocyte monolayers were wounded, palladin was rapidly upregulated along the edge of the wound, coordinate with an increase in actin assembly. Palladin upregulation occurred along a similar rapid time course following injury to the cerebral cortex of adult rats. To explore palladin function more directly, palladin cDNA was transfected into stellate astrocytes, which acquired a spread morphology and prominent actin bundles. These results suggest that palladin upregulation following injury may be a key step in the acquisition of the reactive astrocyte morphology.     

Three-dimensional confocal morphometry reveals structural changes in astrocyte morphology in situ

Neuronal activity and many pathological states in the CNS are accompanied by transient astrocytic swelling, which affects excitability, extrasynaptic transmission, and neuron-glia interactions. By using three-dimensional confocal morphometry (3DCM), we quantified the morphometric parameters of astrocytes in intact tissue. In experiments performed in brain cortex slices from transgenic GFAP/EGFP mice, we applied 3DCM to study the dynamic changes in astrocyte morphology during hypotonic stress. Our morphometric analysis showed that the effect of a 10-min application of hypotonic solution (200 mmol/kg) on the swelling of different cell compartments was dependent on the extent of the swelling of the total astrocyte volume. If the swelling of the whole cell, i.e., soma and processes, was less than approximately 10%, there were no differences between the swelling of the soma and the processes. However, if the swelling of the total cell volume was greater than 10%, the swelling of the processes was greater than the swelling of the soma. Analyzing the effect of hypotonic solution on the morphology of these astrocytes revealed that the total cell volume increased; however, certain cell compartments were distinguished in which the volume increased, whereas in other compartments cell volume decreased or apparently did not change, and the structure of some compartments was altered. Our data show that astrocytes in brain slices undergoing hypotonic stress display cell volume regulation as well as transient changes in morphology.     

Phorbol ester-induced change in astrocyte morphology: correlation with protein kinase C activation and protein phosphorylation

Treatment with 300 nM phorbol 12-myristate 13-acetate (PMA) transforms polygonal-shaped cultured astrocytes into process-bearing cells and produces a shift in protein kinase C (PK-C) from the cytosol to the membrane. Exposure to PMA also produces increases in the phosphorylation of several proteins including vimentin, glial fibrillary acidic protein (GFAP), an acidic 80,000 molecular weight protein, and two 30,000 molecular weight proteins (pI 5.5 and 5.7). The effects of PMA on the translocation of PK-C and on protein phosphorylation precede the PMA-induced changes in astrocyte morphology, and a close correlation exists between the concentration of PMA necessary to elicit half-maximal and maximal effects on the shift of PK-C to the membrane and on protein phosphorylation. In addition, the PMA-induced alterations in cell morphology are not permanent, and within 24 hr after PMA treatment the cells have reverted almost to their original morphology. A second exposure to PMA at this time fails to elicit further change in cell shape and is also incapable of producing increases in the phosphorylation of proteins. It was determined that there is little, if any, PK-C present in these PMA-pretreated cells. The morphological responsiveness to PMA gradually returns in 5 to 8 days after the initial treatment with PMA, and this is accompanied by the recovery of PK-C activity and the phosphorylation response. Therefore, these studies suggest that the effect of PMA on astrocyte morphology is mediated by the activation of PK-C and subsequent protein phosphorylation.     

Sexual differentiation of astrocyte morphology in the developing rat preoptic area

The preoptic area is an important brain region controlling sex-typic behaviour and physiology, and astrocytes of this region are responsive to steroids perinatally. Utilizing glial fibrillary acidic protein immunocytochemistry, the morphology of astrocytes in the preoptic area of male and female rat pups was examined on the day of birth and on postnatal day 3. As early as the day of birth, astrocytes of the male preoptic area exhibit both significantly greater primary process length and number of primary processes, and these differences remain at postnatal day 3. Application of exogenous steroid to females suggested that gonadal steroids, in particular oestradiol, mediate the sex difference. Pups received 100 micro g of steroid on the day of birth and again on postnatal day 1, and astrocyte morphology was assessed on postnatal day 3. Both oestradiol and testosterone induced significant changes in process length and number compared to vehicle-treated controls. Astrocytes of oestradiol-treated females did not differ on PN3 from those of PN3-untreated males. Exposure to the nonaromatizable steroid, dihydrotestosterone, had no effect on any attribute of astrocyte morphology. This suggests the effects induced by testosterone are mediated by oestradiol following local aromatization of the steroid, and not through direct activation of the androgen receptor. Astrocytes are important in synapse formation and efficacy, and we hypothesize a role for astrocyte complexity and differentiation in the establishment of synaptic patterning.     

Molecular form follows function: (un)snaring the SNAREs

Exocytotic release of transmitters is mediated by the ternary SNARE complex. The form of this complex is consistent with its function in the positioning of vesicles to the plasma membrane and their fusion to it. Recent advances in single-molecule techniques, however, bring an additional layer of complexity to this process, implicating that there might be various modes of operation. For example, the binary syntaxin-synaptobrevin 2 complex, in addition to the ternary complex containing SNAP25, might enable vesicular docking. Single-molecule techniques allow direct measurements of the distance/extension, rupture force, spontaneous dissociation times and interaction energy for SNARE protein-protein interactions. These measurements are complementary to results and conclusions drawn from other techniques. Consequently, single-molecule techniques promise tremendous opportunities for in vitro investigations of SNARE proteins to improve our understanding of their role in exocytosis.     

Effects of sphingosine on phorbol ester-mediated changes in astrocyte morphology and protein phosphorylation

Previous studies indicate that phorbol myristate acetate (PMA) can induce morphological changes in astrocytes cultured from the rat neocortex. PMA also increased 32P incorporation into several proteins, including glial fibrillary acidic protein (GFAP), vimentin, and proteins with molecular weights of 80,000 (pI 4.5), 50,000 (pI 4.9), and 30,000 (pI 5.5). The present studies were conducted to determine if the morphological effect and the phosphorylation effect of PMA could be blocked by treatment with sphingosine, a protein kinase C inhibitor. Treatment with 15 microM sphingosine inhibited the effect of PMA on astrocyte morphology. This agent also inhibited the increase in phosphorylation mediated by PMA. The percent inhibition ranged from approximately 20% for the 30,000-Mr protein to 70% for GFAP. Analysis of phosphorylation sites on GFAP and vimentin using two-dimensional tryptic mapping techniques indicate that the partial inhibition of phosphorylation is likely the consequence of partial inhibition of protein kinase C rather than a selective inhibition at some phosphorylation sites and not others. In addition to increasing 32P incorporation into various proteins, PMA also decreased 32P incorporation in several 20,000-Mr proteins (pI values of 6.7, 6.4, 6.2, 4.9). However, this effect was not blocked by treatment with sphingosine. This suggests that the actions of PMA to increase and decrease 32P incorporation are mediated by different mechanisms.     

Direct and indirect effects of corticosteroids on astrocyte function

Corticosteroids are used for a variety of conditions; among the most well-known uses are for asthma and eczema. We review here the direct and indirect effects of corticosteroids on astrocyte physiology. Astrocytes play an important role in communication between neural cells, as one astrocyte can communicate with many neurons. They are also central in bringing nutrients through the blood-brain barrier (BBB) to the brain areas they serve. Therefore, any chemical or pharmaceutical product entering the brain via the BBB will first come into contact with the astrocytes. We discuss the direct effects that corticosteroids have on astrocyte physiology and functioning; these include inhibited glucose transport, decreased glycogen synthesis and decreased glutamate uptake. Furthermore, the indirect effects of corticosteroids on astrocytes are also reviewed. We know that corticosteroids lower neural serotonin. Lowered serotonin affects astrocyte functioning, and particularly astrocytic cAMP activities, a decrease in cytokine activities and impaired GABA uptake. These can be seen as the indirect effects of corticosteroids on astrocyte physiology. Corticosteroids therefore have a pertinent effect on neuro-energetics due to astrocyte physiology impairment, and this may ultimately be the reason for memory impairment of patients who chronically use corticosteroids.     

Form and function in craniofacial deformities

The interplay of form and function is recognized throughout nature. Whether at the cellular level or visible form, physiological function will not be optimal if not supported by ideal morphology. This principle could not be more true than in the relationship between the human skull and face. The development of ideal skull and facial skeletal form is critical for the function of the brain, vision, airway, mastication, and speech. When craniofacial structure is altered by birth defects, proper functioning is drastically affected. We review the neurocranial basis for normal craniofacial skeletal development and present craniofacial abnormalities that illustrate their deleterious affect on facial function.     

异丙酚干预星形胶质细胞形态学变化及水通道蛋白-4的表达

Ammonia induces astrocyte swelling, which is strongly associated with overexpression of aquaporin-4. However, the mechanisms by which ammonia induces astrocyte swelling, and subsequently upregulating aquaporin-4 expression, remain unknown. In the present study, astrocytes were cultured in vitro and exposed to ammonium chloride (NH4Cl), followed by propofol, protein kinase C agonist, or antagonist, respectively. Astrocyte morphology was observed by light microscopy, and aquaporin-4 expression was detected by western blot analysis. Results showed that propofol or protein kinase C agonist significantly attenuated the degree of NH4Cl-induced astrocyte swelling and inhibited increased aquaporin-4 expression. Propofol treatment inhibited aquaporin-4 overexpression in cultured astrocyte induced by NH4Cl; protein kinase C pathway activation is potentially involved.     

Neuronal regulation of astrocyte morphology in vitro is mediated by GABAergic signaling

The addition of isolated neurons to monolayers of cultured astrocytes induced a morphological change in the astrocytes that came into contact with the added neuronal cell bodies or neurites. The change, which included an increase in the complexity of cell shape, took at least 3 days to become detectable and was enhanced in proportion to the number of attached neurons. Astrocytes that did not make contact with any neurons had a less complex contour, comparable to those in control cultures with no neurons added. Treatment of neuron-astrocyte cocultures with a sodium channel blocker, tetrodotoxin, suppressed the neuron-induced morphological changes in astrocytes. A GABA_A-receptor antagonist, bicuculline, mimicked the inhibitory effect of tetrodotoxin. In cultures without added neurons, morphological alteration of astrocytes was also observed when cultures were incubated for 1 or more days with exogenous GABA together with a GABA-uptake inhibitor, 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol. The effect of exogenous GABA was mimicked by treatment with a GABA_A-receptor agonist, muscimol, and blocked by bicuculline treatment. These results suggest that GABA released from neurons with their activity serves as a signal from neurons to astrocytes that triggers the morphological change in astrocytes through the activation of GABA_A receptors. GLIA 20:1-9, 1997. © 1997 Wiley-Liss, Inc.     

Impairment in auditory and visual function follows perinatal viral infection in the rat

Acoustic startle reflexes are elicited by intense tone bursts but inhibited if weak bursts precede reflex elicitation. Rats were infected by intracerebral inoculation with lymphocytic choriomeningitis virus (LCMV) at birth. Compared to control animals, infected animals had higher elicitation and inhibition thresholds and showed recruitment at intense stimulus levels. Histopathology revealed both cochlear and retinal degeneration. Like some infectious agents in humans, perinatal exposure to LCMV in the rat yields a severe polysensory neuropathy.     

Possible function of astrocyte cytochrome P450 in control of xenobiotic phenytoin in the brain: in vitro studies on murine astrocyte primary cultures

[4-(14)C]Phenytoin underwent a rapid cellular uptake by diffusion within 5 min when applied in a concentration of 10 microM to mouse brain astrocyte cultures. Subsequently, a slow linear increase of intracellular radioactivity indicated metabolic trapping of the drug, with final concentrations reaching 144 pmol phenytoin/mg protein in the astrocytes. Phenytoin levels from 1 to 10 microM decreased cell viability by 15%. The action of cytochrome P450 present in astrocytes in concentrations of 16-17 pmol P450/mg protein could explain these slight cytotoxic effects by generating intermediate metabolites of phenytoin. In contrast, concentrations of 50 microM strongly inhibited cell proliferation. A Cyp2c29 immunorelated P450 isoform was expressed in nearly all astrocytes in culture. Intracellular [4-(14)C]phenytoin was degraded to its major metabolites dihydrodiol, p-HPPH, and m-HPPH through a P450-dependent reaction with a specific activity of 0.66 pmol/min x mg protein, or 0.12 pmol/min x mg protein as measured in cell homogenates. These data underscore the importance of astrocytes as brain cells active in the detoxification of foreign substrates, but also in their toxification due to reactive metabolites generated during these metabolic processes. After diffusionary influx of drugs and other xenobiotics, the astrocyte P450 monooxygenases perform an essential role in the mediation of toxicity most frequently encountered in highly vulnerable neurons.     

Modifications in astrocyte morphology and calcium signaling induced by a brain capillary endothelial cell line

Astrocytes extend specialized endfoot processes to perisynaptic and perivascular regions, and thus are positioned to mediate the bidirectional flow of metabolic, ionic, and other transmissive substances between neurons and the blood stream. While mutual structural and functional interactions between neurons and astrocytes have been documented, less is known about the interactions between astrocytes and cerebrovascular cells. For example, although the ability of astrocytes to induce structural and functional changes in endothelial cells is established, the reciprocity of brain endothelial cells to induce changes in astrocytes is undetermined. This issue is addressed in the present study. Changes in primary cultures of neonatal mouse cortical astrocytes were investigated following their coculture with mouse brain capillary endothelial (bEnd3) cells. The presence of bEnd3 cells altered the morphology of astrocytes by transforming them from confluent monolayers into networks of elongated multicellular columns. These columns did not occur when either bEnd3 cells or astrocytes were cocultured with other cell types, suggesting that astrocytes undergo specific morphological consequences when placed in close proximity to brain endothelial cells. In addition to these structural changes, the pharmacological profile of astrocytes was modified by coculture with bEnd3 cells. Astrocytes in the cocultures showed an increased Ca2+ responsiveness to bradykinin and glutamate, but no change in responsiveness to ATP, as compared to controls. Coculturing the astrocytes with a neuronal cell line resulted in increased responsiveness of the glial responses to glutamate but not to bradykinin. These studies indicate that brain endothelial cells induce changes in astrocyte morphology and pharmacology.     

Factors determining the morphology and distribution of astrocytes in the cat retina: a 'contact-spacing' model of astrocyte interaction

The retina provides a valuable opportunity to examine the interaction of astrocytes with neurones and vasculature, in adult tissue and in vivo. We have studied astrocytes in cat retina to delineate the interactions that determine their morphology and distribution. Their morphology varied with their interaction with surrounding cells, from a classic stellate shape to an elongated bipolar form associated with axon bundles. Evidence is presented that the distribution of astrocytes across the retina is determined by their morphology and by a previously unrecognised interaction between astrocytes, which we term 'contact-spacing,' in which astrocytes maintain contact with their neighbours through their processes, but keep their somas apart. Evidence is also presented that astrocytes are not influenced in their distribution by surrounding neurones, and the influence of developmental mechanisms is identified. These observations are summarised in a contact-spacing model of astrocyte distribution, and four predictions of the model are tested. The concentration of astrocytes along axon bundles dispersed when the axons degenerate but not when vessels were prevented from forming. Further, when both axons and vessels were eliminated, the concentrations of astrocytes dispersed and they became stellate in form. Finally, in the retina of the rat, in which astrocytes show no affinity for axons, the distribution of astrocytes is essentially uniform. We suggest that the contact-spacing interaction among astrocytes provides the anatomical basis of a functional glial network extending across the retina and throughout the central nervous system.