Gregory J. Brewer

Isolation and culture of adult rat hippocampal neurons

Inability to culture adult central neurons and the failure of injured neurons to regenerate in the brain could be due to genetic controls or environmental inhibitors. We tested the environmental inhibitor hypothesis by attempting to regenerate adult rat neurons in B27/Neurobasal culture medium, a medium optimized for survival of embryonic neurons. To isolate neurons from their numerous connections, papain was the best of six different proteases screened on slices of hippocampus for survival of isolated cells after 4 days of culture. Use of a density gradient enabled separation of oligodendroglia and some enrichment of neurons and microglia from considerable debris which was inhibitory to sprouting and viability. With these techniques, about 900000 viable neurons were isolated from each hippocampus of any age rat from birth to 24-36 months, near the median mortality. FGF2 was found to enhance viability at least 3-fold to 40-80%, independent of age, without affecting the length of the processes. Neurons were cultured for more than 3 weeks. These methods demonstrate that hippocampal neurons can regenerate axons and dendrites if provided with adequate nutrition and if inhibitors are removed. They also will enable aging studies. Therefore, the concept of environmental growth restriction may be more appropriate for neurons in the brain than the concept of a genetic block that precludes regeneration of processes.

Survival and growth of hippocampal neurons in defined medium at low density: advantages of a sandwich culture technique or low oxygen

The study of development and plasticity of hippocampal circuitry would greatly benefit from methods which allow the long-term culture of neurons at low density under precisely defined culture conditions. We report that isolated hippocampal neurons from embryonic day 18 rats can be cultured for several weeks at low densities which permits the determination of individual connections. A serum-free medium was modified from the formulation of Romijn to include the biological anti-oxidants vitamin E, glutathione, pyruvate, catalase and superoxide dismutase. Neuronal survival of 80% and neuritogenesis greatly exceeded that seen in serum-based cultures. It appeared that vitamins E, A and linolenic acid promoted neuritogenesis. The beneficial effects of the antioxidants suggested a toxic role of oxygen. To directly test this, cultures were incubated in reduced oxygen (9%) and compared to those in the normal 19.7% oxygen (95% air). After 3 days in culture, neurons with processes in 9% oxygen were more than double those in normal oxygen. Neuronal survival and neurite growth could be improved if the cells were grown on a substrate-coated surface covered with a coverslip. Under this condition, cells show a ring of growth between the center and the edge of the coverslip. In 9% oxygen, this ring was closer to the edge of the coverslip than in normal oxygen. The coverslip did not serve as an additional substrate for attachment since it left the neurons attached to the original substrate. However, removal of the coverslip leads to cell death within 24 h, suggesting that the cells had been exposed to a toxic factor. Variations in glial cell content (less than 10%), pH, and pCO2 were demonstrated to be unlikely explanations of the higher survival. These results suggest that growth in a diffusion-limited space, reduction of oxygen concentration to physiological levels and control of toxic oxidation with physiological antioxidants can greatly improve the survival and neuritogenesis of isolated hippocampal neurons in primary culture.

Isolation and culture of adult neurons and neurospheres

Here we present a protocol for extraction and culture of neurons from adult rat or mouse CNS. The method proscribes an optimized protease digestion of slices, control of osmolarity and pH outside the incubator with Hibernate and density gradient separation of neurons from debris. This protocol produces yields of millions of cortical, hippocampal neurons or neurosphere progenitors from each brain. The entire process of neuron isolation and culture takes less than 4 h. With suitable growth factors, adult neuron regeneration of axons and dendrites in culture proceeds over 1–3 weeks to allow controlled studies in pharmacology, electrophysiology, development, regeneration and neurotoxicology. Adult neurospheres can be collected in 1 week as a source of neuroprogenitors ethically preferred over embryonic or fetal sources. This protocol emphasizes two differences between neuron differentiation and neurosphere proliferation: adhesion dependence and the differentiating power of retinyl acetate.

NMDA receptor regulation of neuronal morphology in cultured hippocampal neurons

The relationship of the activity of specific neurotransmitter receptors to neuronal morphology was studied in cultured dentate granule neurons from embryonic rat hippocampus. These experiments were made possible by the development of a defined serum-free culture medium for growth at densities low enough to observe isolated neurons. N-Methyl-D-aspartate (NMDA) and its specific non-competitive antagonist, MK801, affected neuronal morphology in a reciprocal fashion. Like glutamate, NMDA stimulated extensive branching of neuronal processes. Not only was 75% of the branching blocked by MK801, but the longest process was 250% longer in the presence of MK801. These results suggest that neurotransmitters that act on the NMDA receptor may contribute to development and synaptogenesis.

Protective Effect of the Energy Precursor Creatine Against Toxicity of Glutamate and β-Amyloid in Rat Hippocampal Neurons

Abstract: The loss of ATP, which is needed for ionic homeostasis, is an early event in the neurotoxicity of glutamate and β‐amyloid (Aβ). We hypothesize that cells supplemented with the precursor creatine make more phosphocreatine (PCr) and create large energy reserves with consequent neuroprotection against stressors. In serum‐free cultures, glutamate at 0.5‐1 mM was toxic to embryonic hippocampal neurons. Creatine at >0.1 mM greatly reduced glutamate toxicity. Creatine (1 mM) could be added as late as 2 h after glutamate to achieve protection at 24 h. In association with neurotoxic protection by creatine during the first 4 h, PCr levels remained constant, and PCr/ATP ratios increased. Morphologically, creatine protected against glutamate‐induced dendritic pruning. Toxicity in embryonic neurons exposed to Aβ (25‐35) for 48 h was partially prevented by creatine as well. During the first 6 h of treatment with Aβ plus creatine, the molar ratio of PCr/ATP in neurons increased from 15 to 60. Neurons from adult rats were also partially protected from a 24‐h exposure to Aβ (25‐35) by creatine, but protection was reduced in neurons from old animals. These results suggest that fortified energy reserves are able to protect neurons against important cytotoxic agents. The oral availability of creatine may benefit patients with neurodegenerative diseases.

Long-term stability of grafted polyethylene glycol surfaces for use with microstamped substrates in neuronal cell culture

Crucial to long-term stability of neuronal micropatterns is functional retention of the underlying substratum while exposed to cell culture conditions. We report on the ability of covalently bound PEG films in long-term cell culture to continually retard protein adhesion and cell growth. PDMS microstamps were used to create poly-d-lysine (PDL) substrates permissive to cell attachment and growth, and polyethylene glycol (PEG) substrates were used to minimize protein and cell adhesion. Film thickness was measured using null ellipsometry and atomic force microscopy (AFM). Organosilane film structure was examined using Fourier transform infrared (FT-IR) spectroscopy. Long-term film stability in cell culture conditions was tested by immersion in 0.1 M sodium phosphate buffer pH 7.4 for up to one month. Null ellipsometry and water contact measurements indicated that organosilane films were stable up to one month, whereas the PEG film thickness declined rapidly after day 25. Hippocampal cells plated at 200 cells/mm2 on uniform PEG substrates gave a steady increase in biofilm thickness on PEG films throughout the culture, possibly from proteins of neuronal origin. We found that all the layers in the cross-linking procedure were stable in cell culture conditions, with the exception of PEG, which degraded after day 25.

Long-term maintenance of patterns of hippocampal pyramidal cells on substrates of polyethylene glycol and microstamped polylysine

For neurons to attach and remain in precise micropatterns for weeks in culture, background molecules that remain nonpermissive for extended culture durations need to be identified. Nonpermissive background molecules of either polyethylene glycol (PEG) or the amino acid serine (C/sub 3/H/sub 7/NO/sub 3/) were evaluated. The foreground regions were microstamped with 3-, 5-, or 10-/spl mu/m lines of poly-D-lysine (PDL), which promotes neural attachment and growth. After 29 days in culture the foreground compliance, or the fraction of all live somata which rested on the desired PDL surface, averaged 86% for serine and 90% for PEG, with only a small decline. The background compliance, or the fraction of square areas in the pattern background which were free of neurite extension, declined from highs of 40% and 55% (midculture) to 5.5% and 12% (29 days) for serine and PEG, respectively. Images of the cultures suggest that PEG is significantly more effective as a nonpermissive substrate. The authors conclude that these materials, especially PEG, are adequate for the maintenance of long-term patterned cultures of neurons. They believe that this is the first report of high-quality long-term patterning of cultured neurons.

Microcontact Printing for Precise Control of Nerve Cell Growth in Culture

Microcontact printing, facilitated by silane linker chemistry and high-relief stamps, creates precise patterns of proteins, which in turn control growth of hippocampal neurons in culture. This additive, multi-mask technique permits several different molecules to be patterned on the same substrate. The covalent linker technology permits relatively long-term (two-week) compliance of neurons to the stamped pattern against a polyethylene glycol background. When polylysine was stamped adjacent to a laminin/polylysine mixture, neural somata and dendrites preferred the polylysine while axons prefer the mixture or the border between the two.

Apolipoprotein E4 inhibits, and apolipoprotein E3 promotes neurite outgrowth in cultured adult mouse cortical neurons through the low-density lipoprotein receptor-related protein

The apolipoprotein E4 (apoE4) genotype is a major risk factor for Alzheimers disease (AD); however, the mechanism is unknown. We previously demonstrated that apoE isoforms differentially modulated neurite outgrowth in embryonic neurons and in neuronal cell lines. ApoE3 increased neurite outgrowth whereas apoE4 decreased outgrowth, suggesting that apoE4 may directly affect neurons in the brain. In the present study we examined the effects of apoE on neurite outgrowth from cultured adult mouse cortical neurons to examine if adult neurons respond the same way that embryonic cells do. The results from this study demonstrated that (1) cortical neurons derived from adult apoE-gene knockout (apoE KO) mice have significantly shorter neurites than neurons from adult wild-type (WT) mice; (2) incubation of cortical neurons from adult apoE KO mice with human apoE3 increased neurite outgrowth, whereas human apoE4 decreased outgrowth in a dose-dependent fashion; (3) the isoform specific effects were abolished by incubation of the neurons with either receptor associated protein (RAP) or lactoferrin, both of which block the interaction of apoE-containing lipoproteins with the low-density lipoprotein receptor-related protein (LRP). These data suggest a potential mechanism whereby apoE4 may play a role in regenerative failure and accelerate the development of AD.

Modulation of neural network activity by patterning

Using neuronal cultures on microelectrode arrays, researchers have shown that recordable electrical activity can be influenced by chemicals in the culture environment, thus demonstrating potential applicability to biosensors or drug screening. Since practical success requires the design of robust networks with repeatable, reliable responses understanding the sources of variation is important. In this report, we used lithographic technologies to confine neurons to highly defined patterns (40 microm wide stripes); in turn these patterns gave us a measure of control over the local density of neurons (100-500 cells/mm(2)). We found that the apparent electrical activity of the network, as measured by the fraction of electrodes from which signals were recordable, increases 8-10-fold with greater local density. Also, average-firing rates of the active neurons increased 3-5-fold. We conclude that patterned networks offer one means of controlling and enhancing the responsiveness of cultured neural networks.