Glial Contamination Research Articles

Creatine Protects Against Cytosolic Calcium Dysregulation, Mitochondrial Depolarization and Increase of Reactive Oxygen Species Production in Rotenone-Induced Cell Death of Cerebellar Granule Neurons.

Rotenone is a neurotoxin that is an active component of many pesticides which has been shown to induce Parkinsonism in animal models. We show that the cytotoxicity of exposure to nanomolar concentrations of rotenone in cultures of mature cerebellar granule neurons (CGN) in serum-free medium is not due to phagocytosis by glial contamination. A concentration as low as 5.65 ± 0.51nM of rotenone was enough to trigger 50% cell death of mature CGN in culture after 12h. The addition of serum proteins to the culture medium attenuated rotenone neurotoxicity, and this can account at least in part for the requirement of higher rotenone concentrations to elicit neuronal cytotoxicity reported in previous works. Creatine partial protection against CGN death promoted by 5nM rotenone correlated with creatine protection against rotenone-induced mitochondrial depolarization and oxidative stress. Furthermore, creatine largely attenuated the early dysregulation of cytosolic Ca2+ concentration after acute rotenone treatment. Noteworthy, our results also revealed that the sustained alteration of Ca2+ homeostasis induced by rotenone takes place at the onset of the enhancement of intracellular oxidative stress and before mitochondrial depolarization, pointing out that cytosolic Ca2+ dysregulation is a very early event in the rotenone toxicity to CGN.

Long-term primary culture of neurons taken from chick embryo brain: A model to study neural cell biology, synaptogenesis and its dynamic properties

BackgroundStudying neuronal growth, development and synaptogenesis are among the hot research topics. However, it is faced with various challenges and technical limitations that include but not limited to donor's species and health, threat to life, age of embryo, glial contamination, real-time tracking, and follow-up. New methodWe have successfully standardized a method for long-term primary culture of neurons collected from post-fertilized 9 day incubated chicken embryo brain overcoming the limitations mentioned above. Fertilized eggs were incubated in the laboratory and neurons from the embryonic brain were collected and low-density culture, apparently without glial contamination, was studied at least for 35 days in vitro (DIV). ResultsNeurons were characterized by double immunostaining using stringent neuronal and glial markers. Neuronal differentiation, cytomorphology, neurite and axon formation, development and maturation, spine formation and synaptogenesis were tracked in real-time in a stage and time dependent manner. The neurons were transfected with Synaptophysin-RFP to label synaptic vesicles, which were followed in real-time under live-cell imaging. Comparison with existing methodsEvery step was carried out under controlled laboratory conditions. Eggs are easily available, easy to handle, neurons from desired day of incubation could be conveniently studied for long period in apparently glia-free condition. In addition to common factors affecting primary culture, selection of culture media and cover glass coating are other key factors affecting neuronal cultures. ConclusionsWe describe an inexpensive, simpler pure primary neuronal culture method for studying neuronal cell-biology, synaptogenesis, vesicular dynamics and it has potential to grow 3D-multilayered brain in vitro.

Isolation and characterization of human cholinergic neurons from fetal nucleus basalis of Meynert

The nucleus basalis of Meynert (NBM), the major source of cholinergic innervation to the neocortex, is selectively vulnerable to degeneration in Alzheimer’s disease (AD; 1). To date no resolutive therapies exist. Among the therapeutic strategies currently in development, cell transplantation for the replacement of lost neurons is under consideration. The preliminary clinical experiences reported in the literature are limited to a very restricted number of patients and extensive studies are needed to identify the optimal cell source and the most effective methods for cell transplantation in AD. In this study we established a primary culture of human cholinergic neurons (hCN) isolated from the NBM of 12-week old fetuses. The primary culture was immunophenotyped with flow cytometry and resulted almost totally positive for the neuronal marker MAP2 and for the choline acetyltransferase (ChAT; 97±2 %). A small percentage of cells was positive for O4 (4±2.5%) and GFAP (1.6±0.05%), suggesting a marginal glial contamination. ChAT expression in hCN was confirmed by immunocytochemistry and western blot analyses. Moreover, hCN express the nerve growth factor (NGF) receptor TrkA, which is biologically active, as demonstrated by both increased phosphoTrkA detection and cell growth stimulation upon NGF treatment. Interestingly, hCN also express receptors for hormones of the hypothalamicpituitary- gonadal (HPG) axis (ERs, LHR, GnRHR), indicating that our cellular model may be a useful tool to study in vitro the mechanisms underlying the known association between HPG hormones and neurodegeneration (2). To test whether hCN could be used in cell therapy, these cells were intravenously administered in NBM quisqualic acid-lesioned adult male rats. Histological examination of paraffin embedded brain slices from injected rats showed that hCN (labeled with PKH26 fluorescent kit before injection) migrated to the injected NBM and along the needle tract. Behavioral tests indicated a significant improvement in cognitive and spati

Soluble Axoplasm Enriched from Injured CNS Axons Reveals the Early Modulation of the Actin Cytoskeleton

Axon injury and degeneration is a common consequence of diverse neurological conditions including multiple sclerosis, traumatic brain injury and spinal cord injury. The molecular events underlying axon degeneration are poorly understood. We have developed a novel method to enrich for axoplasm from rodent optic nerve and characterised the early events in Wallerian degeneration using an unbiased proteomics screen. Our detergent-free method draws axoplasm into a dehydrated hydrogel of the polymer poly(2-hydroxyethyl methacrylate), which is then recovered using centrifugation. This technique is able to recover axonal proteins and significantly deplete glial contamination as confirmed by immunoblotting. We have used iTRAQ to compare axoplasm-enriched samples from naïve vs injured optic nerves, which has revealed a pronounced modulation of proteins associated with the actin cytoskeleton. To confirm the modulation of the actin cytoskeleton in injured axons we focused on the RhoA pathway. Western blotting revealed an augmentation of RhoA and phosphorylated cofilin in axoplasm-enriched samples from injured optic nerve. To investigate the localisation of these components of the RhoA pathway in injured axons we transected axons of primary hippocampal neurons in vitro. We observed an early modulation of filamentous actin with a concomitant redistribution of phosphorylated cofilin in injured axons. At later time-points, RhoA is found to accumulate in axonal swellings and also colocalises with filamentous actin. The actin cytoskeleton is a known sensor of cell viability across multiple eukaryotes, and our results suggest a similar role for the actin cytoskeleton following axon injury. In agreement with other reports, our data also highlights the role of the RhoA pathway in axon degeneration. These findings highlight a previously unexplored area of axon biology, which may open novel avenues to prevent axon degeneration. Our method for isolating CNS axoplasm also represents a new tool to study axon biology.

Tumor Necrosis Factor-α Potentiates Intraneuronal Ca2+ Signaling via Regulation of the Inositol 1,4,5-Trisphosphate Receptor

Inflammatory events have long been implicated in initiating and/or propagating the pathophysiology associated with a number of neurological diseases. In addition, defects in Ca2+-handling processes, which shape membrane potential, influence gene transcription, and affect neuronal spiking patterns, have also been implicated in disease progression and cognitive decline. The mechanisms underlying the purported interplay that exists between neuroinflammation and Ca2+ homeostasis have yet to be defined. Herein, we describe a novel neuron-intrinsic pathway in which the expression of the type-1 inositol 1,4,5-trisphosphate receptor is regulated by the potent pro-inflammatory cytokine tumor necrosis factor-alpha. Exposure of primary murine neurons to tumor necrosis factor-alpha resulted in significant enhancement of Ca2+ signals downstream of muscarinic and purinergic stimulation. An increase in type-1 inositol 1,4,5-trisphosphate receptor mRNA and protein steady-state levels following cytokine exposure positively correlated with this alteration in Ca2+ homeostasis. Modulation of Ca2+ responses arising from this receptor subtype and its downstream effectors may exact significant consequences on neuronal function and could underlie the compromise in neuronal activity observed in the setting of chronic neuroinflammation, such as that associated with Parkinson disease and Alzheimer disease.

Neuroprotective effects of caffeine against complex I inhibition–induced apoptosis are mediated by inhibition of the Atm/p53/E2F‐1 path in cerebellar granule neurons

The aim of the present study was to evaluate the neuroprotective effects of caffeine, an inhibitor of ataxia telangiectasia mutated (ATM) enzyme and an antagonist of adenosine receptors, in two models of apoptosis in cerebellar granule neurons (CGNs): the inhibition of mitochondrial complex I by the neurotoxin MPP(+) and serum and potassium deprivation. We used cerebellar granule neurons because of low glial contamination. Cell viability was measured by the MTT method, and apoptosis was evaluated by assessing DNA fragmentation with flow cytometry or quantification of nuclear condensation. Our data indicate that the neuroprotective effects of caffeine in the MPP+ model of apoptosis are mediated through activation of the ATM/p53 pathway. In addition, caffeine decreased the expression of cyclin D and the transcription factor E2F-1, a regulator of apoptosis in neurons. Caffeine-mediated neuroprotection was not mediated through blockade of adenosine receptors because DPCPX and CGS-15943, two antagonists of these receptors, failed to attenuate apoptosis produced by MPP+ treatment. In addition, caffeine did not exert neuroprotective effects after serum and potassium withdrawal, a p53-independent model of apoptosis. Taken together, our findings indicate that DNA damage/ATM activation is a key component of MPP+-induced apoptosis in CGNs through activation of p53 and reentry into the cell cycle, specifically expression of the transcription factor E2F-1.

Enhanced induction of the immunoproteasome by interferon gamma in neurons expressing mutant Huntingtin.

Huntington disease (HD) inclusions are stained with anti-ubiquitin and anti-proteasome antibodies. This, together with proteasome activity studies on transfected cell, suggested that alterations in the ubiquitin-proteasome system (UPS) might contribute to HD pathogenesis. In previous work we reported that in a conditional mouse model of Huntington's disease (HD94 mice), the chymiotrypsin- and trypsin-"like" activities of the proteasome are increased selectivity in the affected and aggregate-containing brain regions: striatum, and cortex. Moreover, in these areas a neuronal increase in the interferon-inducible subunits of the immunoproteasome LMP2 and LPM7 was observed. In order to test if the expression of N-terminal mutant huntingtin (htt) by itself is sufficient to induce the change in proteasome catalytic activities as well as in LMP2 subunit expression, we performed activities of the proteasome and western blot experiments in striatal cultured neurons from HD94 mice free of glial contamination. We found no changes in any of the activities in these cells. Furthermore, western blot analysis performed with specific antibody against LMP2 subunits, revealed no difference in levels of this subunit in striatal neurons from HD94 compared to control cultures were treated with interferon-gamma (IFN-gamma) during 72 hours, a clear increase in LMP2 levels was observed in control neuronal cultures. Interestingly, this increase was much more pronounced (95% higher) in HD94 striatal cultures. These results indicate that although expression of mutant htt is not sufficient to induce the changes in proteasome catalytic core observed in HD, it synergizes the changes induced by IFN-gamma. Furthermore, immunocytochemical studies revealed that HD94 striatal neuron expressing high levels of LMP2 subunit showed a pre-apoptotic appearance. These results suggest that the correlation between neuronal induction of the immunoproteasome and neurodegeneration found in HD brains is secondary to inflammatory processes.

Culturing hippocampal neurons

The procedure for making a low density culture of hippocampal neurons has been elaborated by Goslin and Banker. The viability of hippocampal neurons, which are sparsely disseminated on the glass surface, is maintained by a separately cultured glial monolayer; the glial feeder layer is grown on the bottom surface of the dish, while those neurons, placed face down, are attached on the coverslips. This method is originaLly designed for the observation of the maturation, polarity and axogenesis of a single neuron. In addition, this method can be applied for a variety of other purposes: (1) to observe synaptogenesis, (2) to analyze synaptic function electrophysiologically, (3) to analyze receptor functions and signaling cascades pharmacologically, (4) to visualize a molecular dynamics by time-lapse analyses of GFP-tagged molecules, and (5) to observe ultrastructure by an electron microscope. Furthermore, these neurons are useful even in biochemical experiments because they are relatively uniform without glial contamination and highly enriched in synaptic components.

Bax Is Increased in the Retina of Diabetic Subjects and Is Associated with Pericyte Apoptosis in Vivo and in Vitro

Diabetes of even short duration accelerates the death of capillary cells and neurons in the inner retina by a process consistent with apoptosis. We examined whether the process is accompanied by changes in the expression of endogenous regulators of apoptosis. In postmortem retinas of 18 diabetic donors (age 67 +/- 6 years, diabetes duration 9 +/- 4 years) the levels of pro-apoptotic Bax were slightly, but significantly, increased when compared with levels in 20 age-matched nondiabetic donors (P = 0.04). In both groups, Bax localized to vascular and neural cells of the inner retina. Neither pro-apoptotic Bcl-X(S), nor pro-survival Bcl-X(L) appeared affected by diabetes. The levels of these molecules could not be accurately quantitated in lysates of retinal vessels because of variable degrees of glial contamination. However, studies in situ showed in several pericytes, the outer cells of retinal capillaries, intense Bax staining often in conjunction with DNA fragmentation. Bovine retinal pericytes exposed in vitro to high glucose levels for 5 weeks showed elevated levels of Bax (P = 0.03) and increased frequency of annexin V binding, indicative of early apoptosis. Hence, human diabetes selectively alters the expression of Bax in the retina and retinal vascular pericytes at the same time as it causes increased rates of apoptosis. The identical program induced by high glucose in vitro implicates hyperglycemia as a causative factor in vivo, and provides a model for establishing the role of Bax in the accelerated death of retinal cells induced by diabetes.

Survival of Purified Rat PhotoreceptorsIn VitroIs Stimulated Directly by Fibroblast Growth Factor-2

Basic fibroblast growth factor (FGF-2) influences the differentiation and survival of retinal photoreceptors in vivo and in vitro, but it is not known whether it acts directly on photoreceptor FGF receptors or indirectly through activation of surrounding cells. To clarify the effects of FGF-2 on photoreceptor survival, we developed a purified photoreceptor culture system. The outer nuclear layers of postnatal day 5-15 rat retinas were isolated by vibratome sectioning, and the photoreceptor fractions obtained were enzymatically dissociated. Photoreceptors were maintained in monolayer culture for 1 week in a chemically defined medium. Immunocytochemical labeling showed that >99.5% of cells were photoreceptors, and glial contamination represented approximately 0. 2%. Photoreceptors from postnatal day 5-9 retinas survived for at least 24 hr in vitro, whereas cells from postnatal day 10-15 retinas died rapidly. Subsequent studies performed with postnatal day 5 photoreceptors showed that their survival was increased in a dose-dependent manner after the addition of FGF-2. In control cultures, 36% of originally seeded photoreceptors were alive after 5 d in vitro, and in the presence of 20 ng/ml FGF-2 this number was doubled to 62%. This increase was not caused by proliferation of photoreceptor precursors. Denaturing or blocking FGF-2 prevented enhancement of survival. Conversely, only 25.5% of photoreceptors survived in the presence of epidermal growth factor (EGF). FGF- and EGF-receptor mRNA and proteins were detected in purified photoreceptors in vitro, and addition of FGF-2 or EGF led to tyrosine phosphorylation of photoreceptor proteins. These data support a direct mechanism of action for FGF-2 stimulation of photoreceptor survival.

Axonal origin and purity of growth cones isolated from fetal rat brain

The investigation of the molecular properties of nerve growth cones depends to a significant degree on their isolation from fetal brain in the form of ‘growth cone particles’ (GCPs). The availability of markers for developing axons and dendrites, as well as glial cells, has made it possible to characterize the GCP fraction in much greater detail than before and to optimize its yield. Marker analyses show that a member of the N-CAM family (5B4-CAM), synaptophysin, and especially GAP-43 and non-phosphorylated tau, are enriched in the GCP fraction. In contrast, MAP2 and, particularly, glial fibrillary acidic protein and vimentin are fractionated away from GCPs. Furthermore, GCP yield can be doubled relative to the original procedure, without compromising purity, by raising the sucrose concentration of the fractionation gradient's uppermost layer. The results indicate that GCPs are highly purified growth cone fragments with very little glial contamination, and that they are primarily of axonal origin.

Axonal origin and purity of growth cones isolated from fetal rat brain

The investigation of the molecular properties of nerve growth cones depends to a significant degree on their isolation from fetal brain in the form of ‘growth cone particles’ (GCPs). The availability of markers for developing axons and dendrites, as well as glial cells, has made it possible to characterize the GCP fraction in much greater detail than before and to optimize its yield. Marker analyses show that a member of the N-CAM family (5B4-CAM), synaptophysin, and especially GAP-43 and non-phosphorylated tau, are enriched in the GCP fraction. In contrast, MAP2 and, particularly, glial fibrillary acidic protein and vimentin are fractionated away from GCPs. Furthermore, GCP yield can be doubled relative to the original procedure, without compromising purity, by raising the sucrose concentration of the fractionation gradient's uppermost layer. The results indicate that GCPs are highly purified growth cone fragments with very little glial contamination, and that they are primarily of axonal origin.

Medium weight neurofilament mRNA in goldfish Mauthner axoplasm

Although axons are generally considered to lack the ability to synthesize proteins, the Mauthner axon (M-axon) of the goldfish has been reported to contain some of the basic components of the translational machinery, such as transfer RNA (tRNA), ribosomal RNA (rNNA), and ribosomes. To determine if the M-axon also contains mRNA, we isolated samples of M-axoplasm free of glial contamination as demonstrated by the absence of glial-specific mRNA and protein. Reverse transcription-polymerase chain reaction (RT-PCR) of M-axoplasmic cDNA in the presence of primers for the goldfish medium-weight neurofilament (NF-M) gene produced a single product of the expected length for RT-PCR amplification of goldfish NF-M mRNA. This mRNA might direct protein synthesis of NF-M within the M-axoplasm.

Basic and acidic fibroblast growth factors have trophic effects on neurons from multiple CNS regions

Basic fibroblast growth factor (bFGF) supports the survival of neurons from many regions of the E18 fetal rat brain. Survival was significantly increased for neurons derived from the hippocampus, entorhinal cortex (EC), frontal cortex, parietal cortex (PC), occipital cortex, striatum, septum, and thalamus, but not from the subiculum (Sb). The proportion of neurons rescued by bFGF varied among brain regions, suggesting the existence of subpopulations of responsive neurons. Like hippocampal neurons, neurons from the EC and PC required about 1 pM bFGF (10-20 pg/ml) for half-maximal response; striatal neurons, in contrast, required about 3 pM bFGF. Neurite outgrowth after 24 hr exposure was significantly increased for neurons from the hippocampus, EC, and PC, while striatal neurons had only a marginal response. Although bFGF stimulated some astrocytic proliferation in the cultures, glial contamination was maintained at 2% or less. Acidic FGF (aFGF) supported smaller numbers of neurons from each region, although it significantly increased survival of neurons from hippocampus, EC, PC, striatum, and Sb. The concentration required for half-maximal survival was around 100-300 pM (2-5 ng/ml). It appears that bFGF and aFGF are potent trophic factors for many populations of CNS neurons and could potentially play a significant role in nervous system development.

Neurons from chick embryo cerebrum: Ultrastructural and biochemical development in vitro

Primary cultures of neurons prepared from dissociated telencephalon of 8-day-old chick embryos exhibited characteristic differentiated morphology with an interconnecting neurofibrillary network that became increasingly ramified during the 15-day period of culture. All the cells in the culture can be stained positively using the Sevier-Munger silver impregnation technique. The cells and fibers also contain tetanus toxin binding sites and are heavily stained by antitubulin, as visualized by indirect immunofluorescence. Cell processes containing abundant microtubules organized in parallel arrays were observed by immunofluorescence and electron microscopy. Transmission electron microscopy of processes also revealed vesicle-containing boutons with synaptic attachments to other cells. These cultures are free of glial contamination since significant levels of glial markers, glial fibrillary acidic protein or glutamine synthetase activity, were not detected. In addition, cells with the microtubular pattern characteristic of glia or fibroblasts were not observed in typical microscopic fields. Neuronal glutamic acid decarboxylase activity was comparable to that of adult brain while tyrosine hydroxylase and choline acetyltransferase activities were not detectable. Crude membrane preparations from the cultured neurons contain receptors for γ-aminobutyric acid and benzodiazepine at levels which are 30% and 14% those of adult brain. The chick cerebral cultures thus appear enriched in GABAergic neurons. It can be concluded that development of neuron-specific structural and biochemical markers can occur in the absence of glial cells. The culture system therefore provides a useful model for studies of central neurons in vitro.

The characterization of [3H] adenosine uptake into rat cerebral cortical synaptosomes.

Uptake of adenosine, a putative inhibitory transmitter or modulator, was investigated in rat cerebral cortical synaptosomes. The accumulation of [3H] adenosine into synaptosomes, using an adenosine concentration of 10 microM, was linear for 30 min at 30 degrees C. The uptake appeared to be mediated by kinetically saturable processes with apparent Km's of 1 microM ("high-affinity A") and 5 microM ("high-affinity B"), both of which were partially sensitive to the presence of external sodium and calcium ions. Both uptake processes were partially inhibited by 2,4-dinitrophenol, implying the presence of active uptake and diffusional components. A study of the metabolites of adenosine taken up by the two uptake systems indicates that the major metabolites were adenosine and nucleotides. However, adenosine incorporated by the high-affinity A uptake system is more likely to form deaminated metabolites, such as hypoxanthine and inosine, indicating a possible functional difference between the two uptake processes. A detailed comparison of the inhibitory properties of certain adenosine analogues and other pharmacological agents has revealed differences between the two adenosine uptake systems. Since the glial contamination in synaptosomal preparations is well established, one of the uptake systems we observed in the present study might be of glial origin. This notion is supported by the findings that the Km values and kinetic properties of papaverine action in he synaptosomal high-affinity A uptake system are similar to those of astrocytes reported in the literature. In conclusion, the uptake processes of synaptosomal preparations show that accumulation of adenosine into neuronal (and possibly glial) elements may play a major role in regulating the extracellular adenosine concentration. Uptake inhibitors, such as diazepam, may exert, at least in part, their pharmacological actions by interfering with the regulation of extracellular adenosine concentrations.

Bulk separation of neurones and glia: A comparison of techniques

Three different techniques of preparation of neurones and neuropil (glia) from rat cortex, those of Rose, Norton and Poduslo, and Sellinger and Azcurra, were compared according to the proposed criteria of purity, integrity and yield. Morphologically the cells prepared by the Norton and Poduslo and Sellinger and Azcurra methods appeared better and the fractions were purer than Rose method I, though not Rose method II. Based on carbonic anhydrase ratios, the glial contamination of neurones was 11% in Rose cells, 24.8% in Norton and Poduslo cells and 8.3% in Sellinger and Azcurra cells. Lactate dehydrogenase activity was 25% in Sellinger and Azcurra cell suspension and 37% in Norton and Poduslo cell suspension, of that in the Rose suspension. Oxygen consumption and carbon dioxide production in Sellinger and Azcurra suspension was only 15% and in Norton and Poduslo cell suspension 50%, of that in the Rose technique. The metabolic damage observed in the Sellinger and Azcurra preparation could be partially averted by alteration of the disaggregation medium. The relative utility of the techniques is discussed.

Metabolism of putative transmitters in individual neurons of Aplysia californica. Acetylcholinesterase and catechol-O-methyl transferase.

AbstractThe activities of acetylcholinesterase (acetylcholine acetyl‐hydrolase; EC 3.1.1.7) and catechol‐O‐methyl transferase (S‐adenosylmethionine: catechol‐O‐methyl transferase; EC 2.1.1 .a) were measured in the various ganglia of the nervous system of Aplysia californica and in some of the individually identifiable neurons in these ganglia. All of the neurons studied had measurable levels of activity for both enzymes. Since different individual neurons exhibited approximately the same level of activity we concluded that neither of these enzymes could be used to classify neurons as ‘cholinergic’ vs. ‘aminergic’ or ‘cholinoceptive’ vs. ‘aminoceptive'. The ubiquitous distribution of either or both of these enzymes in different single neurons may be related to glial contamination.

Differential metabolism of RNA in neuronal-enriched and glial-enriched fractions of rat cerebrum.

Abstract—Cerebral tissue of rat, disrupted by passage through a custom‐designed tissue press, was diluted to a 10% (w/v) cellular suspension in 10% (w/v) Ficoll and was then fractionated in the Spinco Model L ultracentrifuge into: (1) two enriched cellular layers (alpha and beta) in an isopycnic Ficoll gradient in the Spinco 40 angular rotor; or (2) into four layers (A, B, C, and D) in a discontinuous Ficoll gradient in the Spinco SW 39 swinging bucket rotor. The cellular composition of these layers was identified microscopically and enzymically as either glial‐enriched (alpha and B layers) or neuronal‐enriched (beta and C layers) fractions of cerebral cortex. Portions of the neuronal and glial fractions were used for determinations of total nitrogen, and for colorimetric determinations of carbonic anhydrase activity (a glial cell marker). These data established that glial contamination of the neuronal‐enriched layer averaged 6·5 per cent. The data also indicated glial enrichment of Layer B, although no quantitative assessment of the amount of neuronal contamination was possible.The kinetics of metabolism of RNA in the glial‐enriched and neuronal‐enriched fractions were studied from 0·5 to 16 h after‐intracisternal injection of either [3H]cytidine or [3H]orotic acid. In addition, the incorporation of [3H]cytidine into crude nuclear and cytoplasmic components of the layers was studied by the use of 1 h pulses. Our findings indicated greater incorporation of [3H]cytidine into nuclear fractions than into cytoplasmic fractions at 1 h and greater incorporation of both precursors into neuronal‐enriched fractions than into glial‐enriched fractions at all pulse times.