Astroglial regulation of apolipoprotein E expression in neuronal cells. Implications for Alzheimer's disease

Although apolipoprotein (apo) E is synthesized in the brain primarily by astrocytes, neurons in the central nervous system express apoE, albeit at lower levels than astrocytes, in response to various physiological and pathological conditions, including excitotoxic stress. To investigate how apoE expression is regulated in neurons, we transfected Neuro-2a cells with a 17-kilobase human apoE genomic DNA construct encoding apoE3 or apoE4 along with upstream and downstream regulatory elements. The baseline expression of apoE was low. However, conditioned medium from an astrocytic cell line (C6) or from apoE-null mouse primary astrocytes increased the expression of both isoforms by 3-4-fold at the mRNA level and by 4-10-fold at the protein level. These findings suggest that astrocytes secrete a factor or factors that regulate apoE expression in neuronal cells. The increased expression of apoE was almost completely abolished by incubating neurons with U0126, an inhibitor of extracellular signal-regulated kinase (Erk), suggesting that the Erk pathway controls astroglial regulation of apoE expression in neuronal cells. Human neuronal precursor NT2/D1 cells expressed apoE constitutively; however, after treatment of these cells with retinoic acid to induce differentiation, apoE expression diminished. Cultured mouse primary cortical and hippocampal neurons also expressed low levels of apoE. Astrocyte-conditioned medium rapidly up-regulated apoE expression in fully differentiated NT2 neurons and in cultured mouse primary cortical and hippocampal neurons. Thus, neuronal expression of apoE is regulated by a diffusible factor or factors released from astrocytes, and this regulation depends on the activity of the Erk kinase pathway in neurons.     

Widespread immunoreactivity for neuronal nuclei in cultured human and rodent astrocytes

The monoclonal antibody (mAb) neuronal nuclei (NeuN) labels the nuclei of mature neurons in vivo in vertebrates. NeuN has also been used to define post-mitotic neurons or differentiating neuronal precursors in vitro . In this study, we demonstrate that the NeuN mAb labels the nuclei of astrocytes cultured from fetal and adult human, newborn rat, and embryonic mouse brain tissue. A non-neuronal fibroblast cell line (3T3) also displayed NeuN immunoreactivity. We confirmed that NeuN labels neurons but not astrocytes in sections of P10 rat brain. Western blot analysis of NeuN immunoreactive species revealed a distribution of bands in nucleus-enriched fractions derived from the different cell lines that was similar, but not identical to adult rat brain homogenates. We then examined the hypothesis that the glial fibrillary acidic protein/NeuN-double positive population of cells might correspond to neuronal precursors. Although the NeuN-positive astrocytes were proliferating, no evidence of neurogenesis was detected. Furthermore, expression of additional neuronal precursor markers was not detected. Our results indicate that primary astrocytes derived from mouse, rat, and human brain express NeuN. Our findings are consistent with NeuN being a selective marker of neurons in vivo , but indicate that studies utilizing NeuN-immunoreactivity as a definitive marker of post-mitotic neurons in vitro should be interpreted with caution.     

Astrocytes protect neurons from nitric oxide toxicity by a glutathione-dependent mechanism

Nitric oxide (NO) contributes to neuronal death in cerebral ischemia and other conditions. Astrocytes are anatomically well positioned to shield neurons from NO because astrocyte processes surround most neurons. In this study, the capacity of astrocytes to limit NO neurotoxicity was examined using a cortical co-culture system. Astrocyte-coated dialysis membranes were placed directly on top of neuronal cultures to provide a removable astrocyte layer between the neurons and the culture medium. The utility of this system was tested by comparing neuronal death produced by glutamate, which is rapidly cleared by astrocytes, and N-methyl-D-aspartate (NMDA), which is not. The presence of an astrocyte layer increased the LD(50) for glutamate by approximately four-fold, but had no effect on NMDA toxicity. Astrocyte effects on neuronal death produced by the NO donors S-nitroso-N-acetyl penicillamine and spermine NONOate were examined by placing these compounds into the medium of co-cultures containing either a control astrocyte layer or an astrocyte layer depleted of glutathione by prior exposure to buthionine sulfoximine. Neurons in culture with the glutathione-depleted astrocytes exhibited a two-fold increase in cell death over a range of NO donor concentrations. These findings suggest that astrocytes protect neurons from NO toxicity by a glutathione-dependent mechanism.     

Cryopreservation of embryonic cerebral tissue of rat.

Embryonic cerebral tissues (ECT) either fresh or frozen-stored, were cultured and transplanted into the cerebella of neonatal host rats. Many variables including composition of the freezing medium, freezing and thawing rates, and storage time in liquid nitrogen were studied systematically. The results indicated that the following conditions yielded good results for tissue culture: using 1 M Me2SO as the cryoprotectant, freezing the brain tissues at a rate of 1 degrees C/min until it reached -70 degrees C, storing the frozen samples in liquid nitrogen and thawing them fast in a 37 degrees C water bath. The viability of the frozen-thawed tissues was assessed by their abilities to grow and differentiate in vitro and in vivo after intracerebral grafting. In tissue culture, growth and differentiation were similar to those of the fresh ECT. Cell morphology and staining reactions were normal in supravital methylene blue staining, cresyl violet staining, and acetylcholinesterase staining. Neurons had well-developed Nissl bodies, and cholinergic neurons also differentiated. Autoradiographic studies showed that more than 50% of the neurons had the ability to uptake gamma-aminobutyric acid with high affinity. In brain tissue transplantation, 9 of 12 transplants survived subsequent grafting after cryopreservation. Moreover, the grafts of surviving cryopreserved tissue displayed cytological and cytoarchitectural characteristics identical to those of fresh grafts. All grafts were integrated with the surrounding host neural tissue. This suggested that there may be synaptic connections between the transplants and the host brain tissues. From this and similar studies on the subject by others wer conclude that cryopreservation is a feasible method for storage of embryonic brain tissue to be used later for intracerebral grafting.     

S100B protein is released from rat neonatal neurons, astrocytes, and microglia by in vitro trauma and anti-S100 increases trauma-induced delayed neuronal injury and negates the protective effect of exogenous S100B on neurons

S100B protein is found in brain, has been used as a marker for brain injury and is neurotrophic. Using a well-characterized in vitro model of brain cell trauma, we have previously shown that strain injury causes S100B release from neonatal rat neuronal plus glial cultures and that exogenous S100B reduces delayed post-traumatic neuronal damage even when given at 6 or 24 h post-trauma. The purpose of the current studies was to measure post-traumatic S100B release by specific brain cell types and to examine the effect of an antibody to S100 on post-traumatic delayed (48 h) neuronal injury and the protective effect of exogenous S100B. Neonatal rat cortical cells grown on a deformable elastic membrane were subjected to a strain (stretch) injury produced by a 50 ms displacement of the membrane. S100B was measured with an ELISA kit. Trauma released S100B from pure cultures of astrocytes, microglia, and neurons. Anti-S100 reduced released S100B to below detectable levels, increased delayed neuronal injury in traumatized cells and negated the protective effect of exogenous S100B on injured cells. Heat denatured anti-S100 did not exacerbate injury. These studies provide further evidence for a protective role for S100B following neuronal trauma.     

Cryopreservation of Unfertilized Mouse Oocytes: The Effect of Replacing Sodium with Choline in the Freezing Medium

Although embryo cryopreservation has become commonplace in many species, effective methods are not available for routine freezing of unfertilized eggs. Cryopreservation-induced damage may be caused by the high concentration of sodium ions in conventional freezing media. This study investigates the effect of a newly developed low-sodium choline-based medium (CJ2) on the ability of unfertilized, metaphase II mouse eggs to survive cryopreservation and develop to the blastocyst stagein vitro.Specifically, the effects of cooling to subzero temperatures, thawing rate, LN₂plunge temperature, and equilibration with a low-sodium medium prior to freezing are examined. In contrast to cooling to 23, 0, or −7.0°C in a sodium-based freezing medium (ETFM), cooling in CJ2 had no significant negative effect on oocyte survival or development. Oocytes frozen in CJ2 survived plunging into LN₂from −10, −20, or −33°C at significantly higher rates than oocytes frozen in ETFM. With the protocol used (1.5 M PrOH, 0.1 M sucrose, −0.3 C/min, plunging at −33°C) rapid thawing by direct submersion in 30°C water was more detrimental to oocyte survival than holding in air for 30 or 120 s prior to transfer to water. Equilibration of unfertilized oocytes with a low-sodium medium prior to cryopreservation in CJ2 significantly increased survival and blastocyst development. These results demonstrate that the high concentration of sodium in conventional freezing media is detrimental to oocyte cryopreservation and show that choline is a promising replacement. Reducing the sodium content of the freezing medium to a very low level or eliminating sodium altogether may allow oocytes and other cells to be frozen more effectively.     

Cytokine Regulation of Nerve Growth Factor-Mediated Cholinergic Neurotrophic Activity Synthesized by Astrocytes and Fibroblasts

The neurotrophic activity of astrocytes and fibroblasts and its regulation by various cytokines were investigated. Astrocyte conditioned medium (ACM) enhanced the survival of neurons and the proliferation of astrocytes in embryonic cortical cultures grown in serum-free defined medium. However, these results were not affected by acidic fibroblast growth factor, interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF alpha), and transforming growth factor-beta 1. In contrast, ACM induced choline acetyltransferase expression in septal cholinergic neurons via nerve growth factor (NGF)-dependent and -independent mechanisms. However, neither acidic nor basic fibroblast growth factor is involved in this biological activity in ACM. The cytokines listed above mainly stimulate NGF-mediated cholinergic neurotrophic activity in ACM. A combination of IL-1 beta and TNF alpha significantly enhanced choline acetyltransferase activity in septal neurons co-cultured with astrocytes, and this effect was found to be mediated by NGF produced by activated astrocytes. Effects of astrocytes on GABAergic neurons were also examined. ACM was found to increase glutamate decarboxylase activity in neuronal cultures from septum in the presence of Ara-C. However, the cytokines did not enhance this activity in ACM. Moreover, a combination of IL-1 beta and TNF alpha had no effect on glutamate decarboxylase activity in septal neurons co-cultured with astrocytes. In a final set of experiments, cholinergic neurotrophic activity in skin-derived fibroblast conditioned medium (FCM) was examined. FCM was found to possess biological activity similar to that of ACM on septal neurons grown in serum-free defined medium with Ara-C. The cytokines also enhanced NGF-mediated cholinergic neurotrophic activity in FCM. Astrocytes and fibroblasts were found to possess NGF-type and non-NGF-type cholinergic neurotrophic activity, and various cytokines were found to regulate the NGF-type cholinergic neurotrophic activity in both types of cells. NGF produced by astrocytes and fibroblasts that are activated by cytokines is likely to be important for development and regeneration of NGF-sensitive neurons in the central and peripheral nervous systems.     

Respiration in Primary Cultured Cerebellar Granule Neurons and Cerebral Cortical Neurons

Respiration was measured polarographically in primary cultures enriched with cerebellar granule neurons or cerebral cortical neurons. The basal respiratory rate, measured on the sixth day after culturing, was 12.00 natom equiv. O/mg protein/min for the cortical neurons and 12.70 natom equiv. O/mg protein/min for the granule neurons. Maximal stimulation by 2,4-dinitrophenol produced a 20-40% increase over the basal rate for both neuronal types. Oligomycin inhibited neuronal basal respiration by 45%. These respiratory rates in neurons from primary culture are markedly lower than those measured in astrocytes grown under similar conditions.     

A histological analysis of liver injury in freezing storage

As part of a more extensive study on the use of high subzero freezing for cryopreservation of mammalian livers we have tried to single out the effects of freezing and thawing on tissue damage. We compared the morphology of livers after freezing and thawing with what we considered an optimal high subzero cryopreservation protocol with the morphology of livers preserved under the same thermal conditions and in the same solution in a supercooled state, without freezing. The results show that while hepatocytes survive high subzero cryopreservation, detachment of endothelial cells occurs in every freezing experiment. On the other hand, the endothelial cells in livers that are not frozen are intact. This suggests that endothelial cell damage is caused by freezing and may be an important factor in high subzero freezing cryopreservation of the liver.     

Evolution of <Emphasis Type="Italic">GLUD2</Emphasis> Glutamate Dehydrogenase Allows Expression in Human Cortical Neurons

Human hGDH2 arose via duplication in the apes and driven by positive selection acquired enhanced catalytic ability under conditions inhibitory to its precursor hGDH1 (common to all mammals). To explore the biological advantage provided by the novel enzyme, we studied, by immunohistochemistry (IHC) and immunofluorescence (IF), hGDH1 and hGDH2 expression in the human brain. Studies on human cortical tissue using anti-hGDH1-specific antibody revealed that hGDH1 was expressed in glial cells (astrocytes, oligodendrocytes, and oligodendrocyte precursors) with neurons being devoid of hGDH1 staining. In contrast, an hGDH2-specific antiserum labeled both astrocytes and neurons. Specifically, hGDH2 immunoreactivity was found in the cytoplasm of large neuronal cells within coarse structures resembling mitochondria. These were distributed either in the perikaryon or in the cell periphery. Double immunofluorescence (IF) suggested that the latter represented hGDH2-labeled mitochondria of presynaptic nerve terminals. Hence, hGDH2 evolution bestowed large human neurons with enhanced glutamate metabolizing capacity, thus strengthening cortical excitatory transmission.     

Increasing tPA Activity in Astrocytes Induced by Multipotent Mesenchymal Stromal Cells Facilitate Neurite Outgrowth after Stroke in the Mouse

We demonstrate that tissue plasminogen activator (tPA) and its inhibitors contribute to neurite outgrowth in the central nervous system (CNS) after treatment of stroke with multipotent mesenchymal stromal cells (MSCs). In vivo, administration of MSCs to mice subjected to middle cerebral artery occlusion (MCAo) significantly increased activation of tPA and downregulated PAI-1 levels in the ischemic boundary zone (IBZ) compared with control PBS treated mice, concurrently with increases of myelinated axons and synaptophysin. In vitro, MSCs significantly increased tPA levels and concomitantly reduced plasminogen activator inhibitor 1 (PAI-1) expression in astrocytes under normal and oxygen and glucose deprivation (OGD) conditions. ELISA analysis of conditioned medium revealed that MSCs stimulated astrocytes to secrete tPA. When primary cortical neurons were cultured in the conditioned medium from MSC co-cultured astrocytes, these neurons exhibited a significant increase in neurite outgrowth compared to conditioned medium from astrocytes alone. Blockage of tPA with a neutralizing antibody or knock-down of tPA with siRNA significantly attenuated the effect of the conditioned medium on neurite outgrowth. Addition of recombinant human tPA into cortical neuronal cultures also substantially enhanced neurite outgrowth. Collectively, these in vivo and in vitro data suggest that the MSC mediated increased activation of tPA in astrocytes promotes neurite outgrowth after stroke.     

Neuronal Soluble Factors Differentially Regulate the Expression of the GLT1 and GLAST Glutamate Transporters in Cultured Astroglia

Abstract: The glutamate transporters in the plasma membranes of neural cells secure termination of the glutamatergic synaptic transmission and keep the glutamate levels below toxic concentrations. Astrocytes express two types of glutamate transporters, GLAST (EAAT1) and GLT1 (EAAT2). GLT1 predominates quantitatively and is responsible for most of the glutamate uptake activity in the juvenile and adult brain. However, GLT1 is severely down-regulated in amyotrophic lateral sclerosis, a progressive neurodegenerative disease. Furthermore, selective loss of this transporter occurs in cultured astroglia. Expression of GLAST, but not of GLT1, seems to be regulated via the glutamate receptor signalling. The present study was undertaken to examine whether neuronal factors, other than glutamate, influence the expression of astroglial glutamate transporters. The expression of GLT1 and GLAST was examined in primary cultures of cerebellar granule neurons, cortical neurons, and astrocytes under different experimental conditions, including those that mimic neuron-astrocyte interactions. Pure astroglial cultures expressed only GLAST, whereas astrocytes grown in the presence of neurons expressed both GLAST (at increased levels) and GLT1. The induction of GLT1 protein and its mRNA was reproduced in pure cortical astroglial cultures supplemented with conditioned media from cortical neuronal cultures or from mixed neuron-glia cultures. This treatment did not change the levels of GLAST. These results suggest that soluble neuronal factors differentially regulate the expression of GLT1 and GLAST in cultured astroglia. Further elucidation of the molecular nature of the secreted neuronal factors and corresponding signalling pathways regulating the expression of the astroglial glutamate transporters in vitro may reveal mechanisms important for the understanding and treatment of neurological diseases.     

Production of GABA by cultured hippocampal glial cells

Medium conditioned by cultured hippocampal glial contains an inhibitory factor that can hyperpolarize and suppress neuronal activity. Using biochemistry, electrophysiology, pharmacology, and mass spectrometry, we have identified the inhibitory factor as GABA (gamma-aminobutyric acid). Like GABA, the inhibitory factor increases chloride and potassium currents in neurons, which can be blocked by bicuculline. Mass spectrometry analysis of conditioned medium reveals peaks that are identical to that for GABA. Up to 500 micromolar GABA is found in conditioned medium from glial cultures. No GABA is found in conditioned medium from neuronal cultures. Hippocampal glia make much more GABA than cortical glia or glia from other brain regions. It is not clear how hippocampal glia synthesize GABA. Although they express GAD mRNA and adding glutamate to the culture medium increases the amount of GABA produced, other data suggest that glia do not use GAD to make GABA. Identifying the mechanism(s) by which GABA is produced by hippocampal glia would help clarify its role in modulating neuronal activity in the brain.     

CNF1 improves astrocytic ability to support neuronal growth and differentiation in vitro

Modulation of cerebral Rho GTPases activity in mice brain by intracerebral administration of Cytotoxic Necrotizing Factor 1 (CNF1) leads to enhanced neurotransmission and synaptic plasticity and improves learning and memory. To gain more insight into the interactions between CNF1 and neuronal cells, we used primary neuronal and astrocytic cultures from rat embryonic brain to study CNF1 effects on neuronal differentiation, focusing on dendritic tree growth and synapse formation, which are strictly modulated by Rho GTPases. CNF1 profoundly remodeled the cytoskeleton of hippocampal and cortical neurons, which showed philopodia-like, actin-positive projections, thickened and poorly branched dendrites, and a decrease in synapse number. CNF1 removal, however, restored dendritic tree development and synapse formation, suggesting that the toxin can reversibly block neuronal differentiation. On differentiated neurons, CNF1 had a similar effacing effect on synapses. Therefore, a direct interaction with CNF1 is apparently deleterious for neurons. Since astrocytes play a pivotal role in neuronal differentiation and synaptic regulation, we wondered if the beneficial in vivo effect could be mediated by astrocytes. Primary astrocytes from embryonic cortex were treated with CNF1 for 48 hours and used as a substrate for growing hippocampal neurons. Such neurons showed an increased development of neurites, in respect to age-matched controls, with a wider dendritic tree and a richer content in synapses. In CNF1-exposed astrocytes, the production of interleukin 1β, known to reduce dendrite development and complexity in neuronal cultures, was decreased. These results demonstrate that astrocytes, under the influence of CNF1, increase their supporting activity on neuronal growth and differentiation, possibly related to the diminished levels of interleukin 1β. These observations suggest that the enhanced synaptic plasticity and improved learning and memory described in CNF1-injected mice are probably mediated by astrocytes.     

Intracellular ascorbic acid inhibits transport of glucose by neurons, but not by astrocytes

It has been demonstrated that glutamatergic activity induces ascorbic acid (AA) depletion in astrocytes. Additionally, different data indicate that AA may inhibit glucose accumulation in primary cultures of rat hippocampal neurons. Thus, our hypothesis postulates that AA released from the astrocytes during glutamatergic synaptic activity may inhibit glucose uptake by neurons. We observed that cultured neurons express the sodium-vitamin C cotransporter 2 and the facilitative glucose transporters (GLUT) 1 and 3, however, in hippocampal brain slices GLUT3 was the main transporter detected. Functional activity of GLUTs was confirmed by means of kinetic analysis using 2-deoxy-d-glucose. Therefore, we showed that AA, once accumulated inside the cell, inhibits glucose transport in both cortical and hippocampal neurons in culture. Additionally, we showed that astrocytes are not affected by AA. Using hippocampal slices, we observed that upon blockade of monocarboxylate utilization by alpha-cyano-4-hydroxycinnamate and after glucose deprivation, glucose could rescue neuronal response to electrical stimulation only if AA uptake is prevented. Finally, using a transwell system of separated neuronal and astrocytic cultures, we observed that glutamate can reduce glucose transport in neurons only in presence of AA-loaded astrocytes, suggesting the essential role of astrocyte-released AA in this effect.     

Cryopreservation of brain tissue for primary culture.

Factors affecting recovery of brain cells from cryopreserved cerebral tissues of fetal rats were examined based on yields of viable cells on cell culture. Favorable preservation was obtained with freezing small pieces (less than 1 mm cube) of brain tissues rather than whole tissues or dissociated single cells, and use of 10% dimethylsulfoxide as a cryoprotectant in liquid nitrogen. As for cell preparation procedures, cell survival was improved when tissues were heated at 32 degrees C during papain digestion and centrifugation. Under favorable conditions, the number of brain cells recovered from cryopreserved tissues corresponded to 20-30% of those from fresh control tissues. Immunocytochemical characteristics of cultured neurons, astrocytes, and oligodendrocytes from cryopreserved and fresh tissues were indistinguishable. Semi-quantitive analyses of microtubule-associated protein-2 (MAP-2) and synaptophysin revealed that there was no difference in the amounts of these markers between cultures from both fresh and cryopreserved tissues. These results suggest that most of all cell types including neurons were equally susceptible to the cryopreservation procedures. We concluded that cryopreservation in liquid nitrogen is an effective method for preservation of embryonic brain tissues for later use in cell culture studies.     

Primary neuronal cultures from the brains of late stage Drosophila pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.     

A new method for the preservation of fungus stock cultures by deep-freezing

Recovery of 66 fungus stock cultures including Oomycota, Zygomycota, Ascomycota, Basidiomycota, and mitosporic mycetes were examined after cryopreservation. Almost all the stock cultures remained viable when the mycelia that had grown over the sawdust medium containing 10% glycerol as the cryoprotectant (65% moisture content, W/W) were frozen rapidly at −85°C and then allow to thaw naturally at room temperature. Test stock cultures were preserved for more than 10 years by this preservation method without any programmed precooling and rapid thawing for their cryopreservation. Most of the test fungi could survive for 5 years in medium containing 10% glycerol even after alternate freezing and thawing at intervals of 6 months. When a strain of Flammulina velutipes was tested for mycelial growth rate and productivity of fruit-bodies after cryopreservation for 3 years, the fungus reproduced with its initial capability. These results demonstrate that the sawdust-freezing method using a cryoprotectant is expected to be a reliable and easy preservation method for fungus stock cultures. Received: December 7, 2000 / Accepted: December 19, 2001     

A cryoprotection method that facilitates cutting frozen sections of whole monkey brains for histological and histochemical processing without freezing artifact

Cutting frozen sections of large (greater than 60 cc) blocks of monkey brain using the conventional procedures of infiltration with 30% sucrose as a cryoprotectant before freezing with pulverized dry ice often produces unacceptable levels of freezing artifact (FA) caused by displacement of tissue by ice crystals. Experiments investigating FA utilized perfusion-fixed brains from 46 monkeys and spanned combinations of cryoprotectants (glycerol, sucrose), freezing methods (dry ice or -75 degrees C isopentane), and fixatives (10% formalin, Karnovsky's or Timm's). The effects were evaluated by rating of FA severity in frozen sections of whole monkey brains. Minor FA appears as enlarged capillaries, more serious FA as large vacuoles, and both first appear midway between the periphery and center of the block. Stronger fixatives increased the severity of freezing artifact. The best method for eliminating FA was graded infiltration with up to 20% glycerol and 2% DMSO (in buffer or fixative), followed by rapid freezing in -75 degrees C isopentane. Although using a glycerol-DMSO infiltration before conventional freezing with pulverized dry ice or using conventional sucrose infiltration before freezing in isopentane gave better results than sucrose infiltration and dry-ice freezing, only the combination of glycerol-DMSO infiltration and freezing in isopentane produced consistently excellent results and virtually eliminated freezing artifact. To determine the effect of freezing with dry ice or isopentane on the rate of cooling in large blocks of CNS tissue, thermocouples were embedded in an 80-cc block of albumin-gelatin and frozen with the two methods. The rate of cooling (-3.5 degrees C/min) was twice as fast using isopentane.