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Long‐Term Culture of Hippocampal Neurons

Carlos Vicario‐Abejón¹

¹Centro de Investigaciones Biológicas, Censejo Superior de Investigaciones Cientificas, Madrid, Spain

Publication Name:

Current Protocols in Neuroscience

Unit Number:

Unit 3.2

DOI:

10.1002/0471142301.ns0302s26

Online Posting Date:

May, 2004

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Abstract

In culture, hippocampal cells can develop to express neuronal antigens and acquire mature neuronal morphologies, including axons, complex dendritic trees, and synapses that are electrophysiologically active. This system is suitable for studying neuronal differentiation and other events, such as synaptogenesis. It is also a valuable model for investigating synaptic plasticity and exploring the mechanisms of neuronal degeneration. This unit provides a protocol for culturing neurons prepared from embryonic (E-18) rat or mouse hippocampus, but could also be used to grow neurons from embryonic cortex, olfactory bulb, striatum, or spinal cord. A second method is included for preparing neuronal cultures from embryos with different genotypes, such as those from transgenic mice. Also described is the preparation of polyornithine- and fibronectin-coated coverslips, which are highly adhesive and promote neurite outgrowth, for use in the culture protocols.

Keywords: hippocampus; embryo; culture; rat; mouse

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Unit Introduction
Basic Protocol: Culture of Hippocampal Neurons from Embryonic Day 18 Rat or Mouse
Alternate Protocol: Culture of Hippocampal Neurons from Transgenic Mouse Embryos
Support Protocol: Polyornithine/Fibronectin-Coated Coverslips
Reagents and Solutions
Commentary
Literature Cited
Figures

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Materials

Basic Protocol: Culture of Hippocampal Neurons from Embryonic Day 18 Rat or Mouse

Materials

Pregnant rat (e.g., Sprague-Dawley, Taconic) or mouse, ~18 days pregnant
CO₂, metophane, or ether to anesthetize animal
70% ethanol
Dissection solution (see recipe), ice cold (keep in ice bucket)
Chopping solution (see recipe)
Trypsinization solution (see recipe)
Supplemented DMEM/F-12/N2/10% FBS (see recipe)
Trituration solution (see recipe)
0.2% to 0.4% trypan blue
PBS with antibiotics (see recipe)
Supplemented DMEM/N2/10% FBS (see appendix 2A for DMEM; see recipe for N2 supplements)
1 mM cytosine -d-arabinofuranoside (Ara-C; Sigma)

Surgical instruments:
- Scissors
- Microdissecting scissors
- Curved forceps (medium and small sizes)
- Dumont no. 5 titanium forceps
- Microdissecting scissors with angled blades (Castroviejo style)
Dissecting microscope and optic fiber lights
Neubauer hemacytometer
Inverted microscope
12-mm-diameter polyornithine/fibronectin-coated circular coverslips for 24-well microtiter plate wells (see Support Protocol)

Additional reagents and equipment for tissue culture and for cell counting using a hemacytometer and trypan blue (see appendix 3B)

Alternate Protocol: Culture of Hippocampal Neurons from Transgenic Mouse Embryos

Additional Materials (also see Basic Protocol)

Pregnant mouse bearing transgenic embryos, 16 to 18 days pregnant
DMEM/F-12/N2 (see recipe)
FBS (appendix 2A or Sigma)

Support Protocol: Polyornithine/Fibronectin-Coated Coverslips

Materials

Concentrated HNO₃
15 µg/ml polyornithine (see recipe)
PBS with antibiotics (see recipe)
1 µg/ml fibronectin (see recipe)

12-mm-diameter circular coverslips for 24-well microtiter plate wells (Bellco Glass)
Staining rack (cover-glass staining outfit, Thomas Scientific)
Oven, 225°C
24-well microtiter plates

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Figures

Figure 3.2.1

Dissection of the embryonic rat hippocampus. (A) With the head firmly grasped with a pair of Dumont forceps, the skin and skull are removed using another pair of forceps, beginning from lambda (arrow) and continuing forward to bregma. The tissue surrounding the head (arrowheads) is also removed. (B) The caudal parts of the CNS, such as the cerebellum, pons, and cervical portions of the spinal cord, are then removed. With the ventral aspect of the brain facing up, the two hemispheres are separated by moving the forceps through the midline (arrow). (C) In the next step, the septum and the diencephalic tissue (thalamus and hypothalamus) of one hemisphere are removed (arrows). The olfactory bulb (ob) is indicated. (D) The hippocampus is visible as a slightly thicker portion lining the curved medial edge of the cortex (arrows). (E) The hippocampus is dissected out by making a longitudinal cut through the border and the cortex. This can be done from either the ventral or dorsal side. Finally, the meninges and choroid plexus surrounding the hippocampus are carefully removed.

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Figure 3.2.2

Expression of MAP-2ab, synapsin I, and GAP-43 proteins by hippocampal neurons in culture. E-18 rat hippocampal neurons grown for 3 weeks in culture, fixed with 4% paraformaldehyde, and double-stained for expression of (A) microtubule-associated protein–2ab (MAP-2ab) and (B) synapsin I. (C) Cells from a different culture were stained for the expression of growth-associated protein (GAP-43). They were then incubated with fluorescein- or rhodamine-conjugated secondary antibodies and visualized and photographed through a microscope using appropriate filters. In mature hippocampal neurons, MAP-2ab expression is restricted to the soma and dendrites, synapsin I concentrates in presynaptic buttons, and GAP-43 is preferentially expressed in the axons (Goslin and Banker, 1991; Vicario-Abejón et al., 1995). Note that compared to dendrites, axons are uniform in diameter and have few branches (although branches may increase in terminal field).

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Literature Cited

Literature Cited
	Altman, J. and Bayer, S. 1990. Prolonged sojourn of developing pyramidal cells in the intermediate zone of the hippocampus and their settling in the stratum pyramidale. J. Comp. Neurol. 301:343-364.
	Altman, J. and Bayer, S.A. 1995. Atlas of Prenatal Rat Brain Development. CRC Press, Boca Raton, Fla.
	Amaral, D.G. and Witter, M.P. 1989 The three-dimensional organization of the hippocampal formation: A review of anatomical data. Neuroscience 31:571-591.
	Banker, G. and Goslin, K. 1998. Culturing Nerve Cells, 2nd ed. MIT Press, Cambridge, Mass.
	Bottenstein, J.E. 1985. Growth and differentiation of neural cells in defined media. In Cell Culture in the Neurosciences (J.E. Bottenstein and G. Sato, eds.) pp. 3-43. Plenum, New York.
	Brewer, G.J., Torricelli, J.R., Evege, E.K., and Price, P.J. 1993. Optimized survival of hippocampal neurons in B27-supplemented neurobasal, a new serum-free medium combination. J. Neurosci. Res. 35:567-576.
	Esteban, L.M., Vicario-Abejón, C., Fernández-Salguero, P., Fernández-Medarde, A., Swaminathan, N., Yienger, K., López, E., Malumbres, M., McKay, R., Ward, M., Pellicer, A., and Santos, E. 2001. Targeted genomic disruption of H-ras and N-ras, individually or in combination, reveals the dispensability of both loci for mouse growth and development. Mol. Cell Biol. 21:1444-1452.
	Geppert, M., Goda, Y., Hammer, R.E., Li, C., Rosahl, T.W., Stevens, C.F., and Sudhof, T.C. 1994. Synaptotagmin I: A major Ca²⁺ sensor for transmitter release at a central synapse. Cell 79:717-727.
	Goslin, K., Asmussen, H., and Banker, G. 1998. Rat hippocampal neurons in low-density culture. In Culturing Nerve Cells (G. Banker and K. Goslin, eds.) pp. 339-370. MIT Press, Cambridge, Mass.
	Harrison, R.G. 1907. Observations on the living developing nerve fiber. Anat. Rec. 1:116-118.
	Kaufman, M.H. 1995. The Atlas of Mouse Development. Academic Press, San Diego.
	Laird, P.W., Zijderveld, A., Linders, K., Rudnicki, M.A., Jaenish, R., and Berns, A. 1991. Simplified mammalian DNA isolation procedure. Nucl. Acids Res. 19:4293-4297.
	Okabe, S., Vicario-Abejón, C. Segal, M., and McKay, R.D.G. 1998. Survival and synaptogenesis of hippocampal neurons without NMDA receptor function in culture. Eur. J. Neurosci. 10:2192-2198.
	Mattson, M.P., Lee, R.E., Adams, M.E., Guthrie, P.B., and Kater, S.B. 1988. Interactions between entorhinal axons and target hippocampal neurons: A role for glutamate in the development of hippocampal circuitry. Neuron 1:865-876.
	Papa, M., Bundman, M.C., Greenberger, V., and Segal, M. 1995. Morphological analysis of dendritic spine development in primary cultures of hippocampal neurons. J. Neurosci. 15:1-11.
	Schlessinger, A.R., Cowan, W.M., and Gottlieb, D.I. 1975. An autoradiographic study of the time of origin and the pattern of granule cell migration in the dentate gyrus of the rat. J. Comp. Neurol. 159:149-176.
	Soriano, E., DelRío, J.A., Martínez, A., and Super, H. 1994. Organization of the embryonic and early postnatal murine hippocampus. I. Immunocytochemical characterization of neuronal populations in the subplate and marginal zone. J. Comp. Neurol. 342:571-595.
	Super, H. and Soriano, E. 1994. The organization of the embryonic and early postnatal murine hippocampus. II. Development of entorhinal, commissural, and septal connections studied with the lipophilic tracer Dil. J. Comp. Neurol. 344:101-120.
	Vicario, C., Tabernero, A., and Medina, J.M. 1993. Regulation of lactate metabolism by albumin in rat neurons and astrocytes from primary culture. Ped. Res. 34:709-715.
	Vicario-Abejón, C., Johe, K.K., Hazel, T.G., Collazo, D., and McKay, R.D.G. 1995. Functions of basic fibroblast growth factor and neurotrophins in the differentiation of hippocampal neurons. Neuron 15:105-114.
	Vicario-Abejón, C., Collin, C., McKay, R.D.G., and Segal, M. 1998. Neurotrophins induce formation of functional excitatory and inhibitory synapses between cultured hippocampal neurons. J. Neursci. 18:7256-7271.
Key Reference
	Banker and Goslin, 1998. See above.
Chapters 2, 3, and 5 discuss general principles for culturing neuronal cells, and Chapter 13 describes a protocol for culturing rat hippocampal neurons.