Electrophysiology of embryonic, adult and aged rat hippocampal neurons in serum-free culture

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Abstract

Methods were recently developed for culturing neurons from adult rat hippocampus using the serum-free medium Neurobasal with B27 supplement. To determine whether adult cultured neurons have normal electrical properties, we studied cultures from rats of three age groups: (1) embryonic; (2) 10–11 months old and (3) 35–36 months old. Neurons had a polarized morphology with a large branching apical dendrite and small basal dendrites. Mean resting potentials were similar in the three age groups. All neurons had nonlinear current-voltage relationships, indicating the presence of voltage-sensitive ion channels. Most neurons had a voltage-sensitive inward current followed by a sustained voltage-sensitive outward current. Tetrodotoxin blocked the inward current, which is likely to be a sodium current. The sustained outward current, which is likely to be a potassium current, reversed at −71 mV. Most neurons exhibited anomalous rectification. Calcium currents were present in both embryonic and adult neurons. Embryonic neurons would sometimes fire multiple action potentials but adult neurons fired only single action potentials. Our results indicate that both embryonic and adult cultured neurons retain a clearly neuronal electrophysiological phenotype in Neurobasal/B27 serum-free medium.

Introduction

In vitro primary culture has been a useful tool for the study of neurons. The method allows for visualization and study of individual neurons, with easy and rapid application of drugs or other substances to the neurons. But primary neuronal culture has suffered from at least three serious limitations. The first limitation is that most culture systems require serum. Serum composition varies from lot to lot, so variability occurs in the culture environment. Concentration differences cause some of this variability, but some is attributable to unknown factors in the serum (reviewed in Banker and Goslin, 1991). A second limitation is that most culture systems work best for embryonic or early postnatal neurons. Neurons from adult animals have been either difficult or impossible to culture (Heuttner and Baughman, 1986, Kaneda et al., 1988). This seriously limits the usefulness of in vitro neuronal culture for questions related to maturation or aging. A third limitation of many primary neuronal culture systems is the presence of astrocytes. Astrocytes are suspected of having many effects on neurons and in mixed glial/neuronal cultures, it is difficult to discern the respective roles of glia and neurons (Mennerick and Zorumski, 1994).

Recently, a new method for primary neuronal culture has been developed using Neurobasal with B27 supplement (Brewer et al., 1993). Neurobasal is a completely defined serum-free medium similar to Dubecco’s modified essential medium and F12. B27 is defined nutritional supplement with essential fatty acids, hormones, vitamins and antioxidants. Together, Neurobasal and B27 are a completely defined medium that can reduce variability in neuronal cultures. Neurobasal/B27 has been systematically tested and each component’s concentration adjusted to produce maximum survival and outgrowth of cultured embryonic hippocampal neurons. Neurobasal/B27 allows survival of approximately 60% of adult and 100% of embryonic neurons after 4 days in culture. Although its composition was optimized using embryonic neurons, Neurobasal/B27 supports growth and survival of adult neurons in culture as well (Brewer, 1997). Neuronal survival and growth is optimized, but survival and growth of glia is attenuated in Neurobasal/B27. In embryonic cultures, over 99% of surviving cells stain for neuronal antigens (Brewer et al., 1993). In adult cultures, 60–70% of surviving cells are neurons, as indicated by staining for neurofilament (Brewer, 1997). Neurobasal/B27 has three potential advantages over previous culture media—absence of serum, attenuated growth of glia and ability to culture adult as well as embryonic or postnatal neurons.

Although Neurobasal/B27 has potential advantages over previous culture methods, the medium has been developed by optimizing components for maximum neuronal survival and growth. It is possible that the culture medium could cause changes in neuronal phenotype that make them unsuitable for use as model neurons. This is perhaps especially true for adult neurons, which previously have been difficult or impossible to culture. Brewer (1997)has performed studies showing that adult neurons cultured in Neurobasal/B27 have a neuronal morphology and stain for neuronal antigens such as tau and neurofilament. This suggests that the cultured neurons have the typical cytoskeletal components of neurons. But an important part of neuronal function involves the passage of ionic currents, with consequent changes in membrane potential that are important in integrating inputs and generating outputs. The purpose of this present study was to determine whether neurons from embryonic (E18), adult (10–11 months old) and aged (35–36 months old) rats cultured in Neurobasal/B27 retained neuron-like electrophysiological properties. Some of these data have been previously reported in abstract form Evans and Brewer (1996).

Section snippets

Culture method

The method of culturing embryonic and adult rat hippocampal neurons is detailed elsewhere (Brewer et al., 1993, Brewer, 1997) and given briefly here. For embryonic neurons, timed-pregnancy mother Sprague-Dawley rats were obtained (Harlan, Indianapolis). On day 18 of gestation, the mother was anesthetized with pentobarbital and the embryos delivered by cesarean section. The embryos were decapitated and the hippocampi removed and placed into Hank’s balanced salt solution with 1 mM pyruvate.

Cell selection

We did not select specific areas of hippocampus for isolation and culture. Since survival is ≈80%, the neuronal population in our cultures may be heterogeneous and include hippocampal areas CA1 and CA3 pyramidal neurons, dentate gyrus granule cells and interneurons. In addition, some glial cells survive in culture. In embryonic cultures, glia constitute less than 1% of the total number of cells. In adult cultures, glial cells (primarily microglia), constitute 30–40% of the cells surviving at 4

Neurons in culture retain a neuronal electrophysiological phenotype

We found that the serum-free growth medium Neurobasal/B27 supports survival and growth of embryonic, adult and aged adult rat hippocampal neurons in primary culture. The cells studied appeared to be neurons by morphological and electrophysiological criteria. Electrophysiologically, most cells had a fast transient tetrodotoxin-sensitive inward current and fired action potentials, which is characteristic of neurons. In this culture system glia constitute a minority of the cells, but a few are

Acknowledgements

The authors thank John Torricelli for preparing cell cultures and Life Technologies for supplying culture media. This work was supported by NINDS grants K08 NS01503 and R29 NS34564 to MSE and an Alzheimer’s Association/Estate of Ruby Hodges Pilot Research Grant to G.J.B.

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