The development of glial fibrillary acidic protein-positive cells and the appearance of laminin-like immunoreactivity in fetal olfactory bulb transplants

Embryonic rat olfactory bulbs were transplanted into the site vacated by aspiration of an olfactory bulb from a neonatal rat. This paper presents our findings related to the development of glial fibrillary acidic protein (GFAP) positive (+ve) glial cells and the appearance of laminin-like immunoreactivity in these transplants. The GFAP + ve glial cells formed perivascular end-feet on the invading vasculature and formed a glia limitans along the glia surface of the transplant. This reconstituted glia limitans was continuous with that of the host brain, there being no glia limitans at the donor-host interface. Thus, the donor tissue was well-integrated with that of the host brain.     

Maintenance and synthesis of proteins for an anucleate axon

The anucleate (distal) segment of a crayfish medial giant axon (MGA) remains intact for months in vivo after severing the axon from its cell body, a phenomenon referred to as long-term survival (LTS). We collected axoplasm from chronic anucleate MGAs by perfusing 2-cm lengths of axons with an intracellular saline. This axoperfusate was analyzed by SDS-PAGE and silver stained. Axoperfusate proteins from intact MGAs and from chronic anucleate MGAs exhibiting LTS for up to 6 months were the same. Furthermore, immunoreactive levels of actin and β-tubulin were similar in axoperfusates from intact and chronic anucleate MGAs. This maintenance of proteins in chronic anucleate MGAs must be due to a lack of protein degradation and/or to local protein synthesis by a source other than the cell body. To investigate local protein synthesis in vitro, we added [³⁵S]-methionine to the extracellular saline surrounding intact and chronic anucleate MGAs. After 4- to 6-h incubations, radiolabelled proteins were detected in axoperfusates analyzed by SDS-PAGE and fluorography. The similarity between radiolabelled proteins in axoperfusates and MGA glial sheaths indicated a glial origin for the radiolabelled axoperfusate proteins. Various observations and control experiments suggested that glial-axonal protein transfer occurred by a physiological process. Glial-axonal protein transfer may contribute to the maintenance of proteins during LTS of chronic anucleate MGAs.     

Ischemic disruption of glutamate homeostasis in brain: quantitative immunocytochemical analyses

More than 10 years ago, it was shown by microdialysis that the excitatory transmitter glutamate accumulates in the interstitial space of brain subjected to ischemic insult. This was one of the key observations leading to the formulation of the `glutamate hypothesis' of ischemic cell death. It is now assumed that even a transient glutamate overflow may set in motion a number of events that ultimately cause cell loss in vulnerable neuronal populations. The aim of the present review is to discuss the intracellular changes that underlie the dysregulation of extracellular glutamate during and after ischemia, with emphasis on data obtained by postembedding, electron microscopic immunogold cytochemistry. While the time resolution of this approach is necessarily limited, it can reveal, quantitatively and at a high level of spatial resolution, how the intracellular pools of glutamate and metabolically related amino acids are perturbed during and after an ischemic insult. Moreover, this can be done in animals whose extracellular amino acid levels are monitored by microdialysis, allowing a direct correlation of extra- and intracellular changes. Immunogold analyses of brains subjected to ischemia have identified dendrites and neuronal somata as likely sources of glutamate efflux, probably mediated by reversal of glutamate uptake. The vesicular glutamate pool has been found to be largely unchanged after 20 min of ischemia. Ischemia causes an increased glutamate content and an increased glutamate/glutamine ratio in glial cells, as revealed by double immunogold labelling. This argues against the idea that glial cells contribute to the extracellular overflow of glutamate in the ischemic brain.     

Distribution of glutamine-like immunoreactivity in the cerebellum of rat and baboon (Papio anubis) with reference to the issue of metabolic compartmentation

Summary The cellular and subcellular localization of glutamine, a major glutamate precursor, was studied by means of an antiserum raised against glutaraldehydefixed glutamine. Ultrathin sections from the cerebellar cortex of rat and baboon (Papio anubis) were incubated sequentially in the primary antiserum and in a secondary antibody coupled to colloidal gold particles. The labelling intensity was quantified by computer-aided calculation of gold particle densities. High levels of immunoreactivity occurred in glial cells (Bergmann fibres, astrocytes, and oligodendrocytes), intermediate levels in cell bodies and processes of granule cells, and low levels in terminals of presumed GABAergic or glutamatergic fibres (terminals of basket and Golgi cells, and of parallel, mossy, and climbing fibres). The labelling intensity of Purkinje cells showed some variation, but never exceeded that in glial cells. Within the nerve fibre terminals, the glutamine-like immunoreactivity showed some preference for mitochondria, but was otherwise evenly distributed. The predominant glial localization of glutamine was also obvious in light microscopic preparations processed according to the postembedding peroxidase-antiperoxidase procedure. Gold particle densities over different types of profile in glutamine immunolabelled sections were compared with particle densities over the corresponding types of profiles in neighbouring sections labelled with an antiserum to glutaraldehyde-fixed glutamate. The glutamate/glutamine ratio, expressed arbitrarily by the ratio between the respective gold particle densities, varied by a factor of about 6, with the highest ratio in the putative glutamatergic mossy and parallel fibre terminals, and the lowest ratio in glial elements. The remaining tissue components displayed intermediate ratios. The present study provides direct morphological evidence for the existence in the brain of distinct compartments with differing glutamate/glutamine ratios.This paper is dedicated to Professor Fred Walberg on the occasion of his 70th birthdayOn leave of absence from Department of Anatomy, Capital Institute of Medicine, You An Men Street, Beijing, China     

Intracellular recordings from isolated rabbit retinal Müller (glial) cells

Müller (glial) cells were isolated from rabbit retinae by papaine and mechanical dissociation. The cells were fixed on a gelatine-covered glass slide by means of concanavalin A, and the slide was mounted in a perfusion chamber under a light microscope with modified optics. Besides the recording microelectrode, two other micropipettes could be adjusted with their tips near the cell. These micropipettes were used for application of test solutions into the environment of the cells. On application of high K⁺ solutions, the cell depolarized strongly but during prolonged application there was a marked repolarization. After the end of high K⁺ application the cells showed a hyperpolarization which was enhanced in both amplitude and duration with prolongation of the K⁺ exposure. Both repolarization and afterhyper-polarization disappeared under ouabain. Ouabain application itself caused a small reversible depolarization. Na⁺ free solution caused hyperpolarization. The results suggest the existence of an active membrane pump mechanism in our cells. This pump seems to be electrogenic under our experimental conditions and seems to be activated even in the absence of sodium. The cell membrane is demonstrated to contain a significant Na⁺ conductance.     

Extracellular ionic and volume changes: The role in glia—Neuron interaction

Activity-related changes in extracellular K⁺ concentration ([K⁺]_e), pH (pH_e) and extracellular volume were studied by means ofion-selective microelectrodes in the adult rat spinal cord in vivo and in neonatal rat spinal cords isolated from pups 3–14 days of age (P3–P14). Concomitantly with the ionic changes, the extracellular space (ECS) volume fraction (α), ECS tortuosity (λ) and non-specific uptake (k′), three parameters affecting the diffusion of substances in nervous tissue, were studied in the rat spinal cord gray matter. In adult rats, repetitive electrical nerve stimulation (10–100 Hz) elicited increases in [K⁺]_e of about 2.0–3.5 mm, followed by a post-stimulation K⁺-undershoot and triphasic alkaline-acid-alkaline changes in pH_e with a dominating acid shift. The ECS volume in the adult rat occupies about 20% of the tissue, α = 0.20 ± 0.003, λ = 1.62 ± 0.02 and k′ = 4.6 ± 0.4 × 10⁻³s⁻¹ (n = 39). In contrast, in pups at P3–P6, the [K⁺]_e increased by as much as 6.5 mm at a stimulation frequency of 10 Hz, i.e. K⁺ ceiling level was elevated, and there was a dominating alkaline shift. An increase in [K⁺]_e as large as 1.3–2.5 mm accompanied by an alkaline shift was evoked by a single electrical stimulus. The K⁺ ceiling level and alkaline shifts decreased with age, while an acid shift, which was preceded by a small initial alkaline shift, appeared in the second postnatal week. In pups at P1–P2, the spinal cord was X-irradiated to block gliogenesis. The typical decrease in [K⁺]_e ceiling level and the development of the acid shift in pH_e at P10–P14 were blocked by X-irradiation. Concomitantly, continuous development of glial fibrillary acidic protein positive reaction was disrupted and densely stained astrocytes in gray matter at P10–P14 revealed astrogliosis.The alkaline, but not the acid, shift was blocked by Mg²⁺ and picrotoxin (10⁻⁶m). Acetazolamide enhanced the alkaline but blocked the acid shift. Furthermore, the acid shift was blocked, and the alkaline shift enhanced, by Ba²⁺, amiloride and SITS. Application of glutamate or gamma-aminobutyric acid evoked an alkaline shift in the pH_e baseline at P3–P14 as well as after X-irradiation. The results suggest that the activity-related acid shifts in pH_e are related to membrane transport processes in mature glia, while the alkaline shifts have a postsynaptic origin and are due to activation of ligand-gated ion channels.At P4–P6, the ECS volume was almost double that in adult rats, α = 0.37 ± 0.01 (n = 17), the ECS tortuosity was significantly higher, λ = 1.78 ± 0.02, while the non-specific uptake was not significantly different, k′ = 3.61 ± 0.56 × 10⁻³ s⁻¹. The α gradually decreased to about 24% at P12. In adult rats, electrical or adequate stimulation evoked a shrinkage of the extracellular space by 20–50%, while no significant changes in ECS volume were found in P3–P6. We conclude that the [K⁺]_e ceiling level, character of the pH_e transients, the size of the ECS volume and the activity-related ECS shrinkage are closely related to gliogenesis.     

Trans-inhibition of glutamate transport prevents excitatory amino acid-induced glycolysis in astrocytes

Previous studies have demonstrated that activation of glutamate transporters promotes glycolysis in astrocytes. Current evidence indicates that compounds such as threo-beta-hydroxyaspartate (THA) are both competitive inhibitors and substrates for glutamate transporters. In this study, we have analyzed the effect of THA on excitatory amino acid (EAA) transport and on EAA-induced glycolysis in mouse primary astrocyte cultures. In agreement with previous studies in rat astrocytes, THA competitively inhibited 3H-D-aspartate (3H-D-Asp) uptake with an IC50 of 319 microM (Ki = 36.6 microM). In contrast, it did not prevent D-aspartate-induced 3H-2-deoxyglucose (2DG) uptake in these conditions. Preexposure of cells to THA for at least 15 min revealed another form of glutamate transport inhibition. This effect was concentration-dependent with an apparent IC50 of 47.7 microM and showed kinetic characteristics consistent with a mechanism of trans-inhibition. Preincubation with THA also inhibited D-aspartate-induced 3H-2DG uptake in a concentration-dependent manner with an apparent IC50 of 59.8 microM. Comparison with other transportable analogues reveals that they share with THA the ability to cause trans-inhibition of glutamate transport and to prevent glutamate-stimulated glycolysis; THA, however, is unique in that it has no effect alone on glucose utilization after preexposure. These data indicate that trans-inhibition of glutamate transport may be a mechanism by which certain glutamate transport inhibitors can prevent the stimulation of aerobic glycolysis by glutamate in astrocytes.     

Immunohistochemical examination of the INK4 and Cip inhibitors in the rat neonatal cerebellum: cellular localization and the impact of protein calorie malnutrition

Expression of the cyclin-dependent kinase inhibitors (CKIs) has been linked to the inhibition of cellular proliferation and the induction of differentiation. Based on structure function analysis, two distinct families of CDKIs, the INK4 and the Cip/Kip family have been identified. The INK4 family member p16(Ink4), and the Cip/Kip protein p27(Kip1) have been implicated in normal development of the CNS and cerebellum. Recent studies have suggested a functional inter-dependence between the CKI and the abundance of cyclin D1 in orchestrating growth factor-induced cellular proliferation. The neonatal rat cerebellum undergoes proliferative growth and differentiation, localized to distinct topographical regions and cell types. The cell type and the temporal profile of CKI expression during postnatal cerebellar development had not been described. The current studies determined the specific cerebellar cell types in which the CKIs were expressed during post natal development by co-staining for cell-type specific markers. p16(Ink4a) and p27(Kip1) immunostaining was identified in both neurons and glial cells, increasing progressively between postnatal days 6 to 13 into adulthood. By contrast, neuronal and glial cell p21(Cip1) staining was prominent at days 6-11 and decreased thereafter. Cyclin D1 was expressed in the proliferating external granular cells, with occassional staining in the molecular, and internal granular layers. Dual immunostaining demonstrated cyclin D1 within cells expressing CKI (p16(Ink4a), p21(Cip1),p27(Kip1)). Cerebellar cellular growth arrest, induced by protein-calorie malnutrition, inhibited cyclin D1 protein levels without affecting CKI immunostaining suggesting CKI do not mediate the developmental arrest. These results demonstrate that the CKIs are induced by differentiation cues in specific cell types with distinct kinetics in the developing cerebellum in vivo.     

Truncated TrkB mediates the endocytosis and release of BDNF and neurotrophin-4/5 by rat astrocytes and schwann cells in vitro

Binding and cross-linking studies with radiolabeled neurotrophins demonstrate that cultured rat hippocampal astrocytes lack full-length TrkB, but do express high levels of truncated TrkB (tTrkB). In astrocytes and Schwann cells, tTrkB appears to have the novel function of mediating the endocytosis of neurotrophins into an acid-stable, Triton X-100 resistant intracellular pool that is released back into the medium in a temperature-dependent manner. Chloroquine treatment, trichloroacetic acid solubility, and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that when incubated with astrocytes or Schwann cells for at least 48 h neither the intracellular nor the released neurotrophins were significantly degraded. The endocytosis and release of neurotrophins may represent a novel mechanism whereby neuroglia can regulate the local concentration of these neurotrophic factors for extended periods of time.