Analysis of Dendritic Spine Morphology in Cultured CNS Neurons
Summary
Numerous recent studies have identified mutations in synaptic proteins associated with brain pathologies. Primary cultured cortical neurons offer great flexibility in examining the effects of these disease-associated proteins on dendritic spine morphology and motility.
Abstract
Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic compartment of synapses. These structures are rich in actin and have been shown to be highly dynamic. In response to classical Hebbian plasticity as well as neuromodulatory signals, dendritic spines can change shape and number, which is thought to be critical for the refinement of neural circuits and the processing and storage of information within the brain. Within dendritic spines, a complex network of proteins link extracellular signals with the actin cyctoskeleton allowing for control of dendritic spine morphology and number. Neuropathological studies have demonstrated that a number of disease states, ranging from schizophrenia to autism spectrum disorders, display abnormal dendritic spine morphology or numbers. Moreover, recent genetic studies have identified mutations in numerous genes that encode synaptic proteins, leading to suggestions that these proteins may contribute to aberrant spine plasticity that, in part, underlie the pathophysiology of these disorders. In order to study the potential role of these proteins in controlling dendritic spine morphologies/number, the use of cultured cortical neurons offers several advantages. Firstly, this system allows for high-resolution imaging of dendritic spines in fixed cells as well as time-lapse imaging of live cells. Secondly, this in vitro system allows for easy manipulation of protein function by expression of mutant proteins, knockdown by shRNA constructs, or pharmacological treatments. These techniques allow researchers to begin to dissect the role of disease-associated proteins and to predict how mutations of these proteins may function in vivo.
Protocol
Discussion
The techniques described above for the detailed quantitative analysis of dendritic spine morphology, linear density and motility in either fixed or live primary cortical neurons are focused on understanding the effects of post-synaptic mechanisms that may contribute to neuropathologies. A similar approach can be used to quantify spine morphology or motility in any spiny neuron, including hippocampal pyramidal, Purkinje, or medium spiny neurons.
The protocol described here can be adapted to lo…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank Kelly Jones for careful editing. This work was supported by NIH grant R01MH 071316, Alzheimer’s Association, the National Alliance for Research on Schizophrenia and Depression (NARSAD), and the National Alliance for Autism Research (NAAR) (P.P.); American Heart Association Postdoctoral Fellowship (D.P.S.); American Heart Association Predoctoral Fellowship (K.M.W.).
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
|---|---|---|---|
| 18 mm round Cover glass No. 1.5 | Warner Instruments | 64-0714 (CS-18R15) | |
| 22 mm square Cover glass No. 1.5 | Warner Instruments | 64-0721 (CS-22S15) | |
| Poly-D-Lysine | Sigma | P-0899 | MW 70~150 Kda |
| Neurobasal Media | Invitrogen | 21103049 | |
| B27 | Invitrogen | 17504044 | |
| Glutamine | Invitrogen | 21051024 | |
| Penicillin-Streptomycin | Invitrogen | 15140148 | |
| D,L-APV (AP-5) | Ascent Scientific | Asc-004 | |
| Lipofectamine 2000 | Invitrogen | 11668019 | |
| DMEM | Invitrogen | 11965092 | |
| HEPES | MediaTech Cellgro | 25-060-C 1 | 1M, pH 7 |
| Formaldehyde Solution | EMD Chemicals | FX0415-5 | 36%, Histology grade |
| Normal Goats Serum | VWR | 100188-514 | Jackson Immunoresearch Labs |
| Triton X-100 | Fisher Scientific | AC21568-2500 | Acros Organics |
| Alexa Fluor 488 goat anti-mouse IgG (H+L) highly cross-adsorbed | Invitrogen | A-11029 | |
| Alexa Fluor 488 goat anti-rabbit IgG (H+L) *highly cross-adsorbed* | Invitrogen | A-11034 | |
| ProLong Gold antifade reagent | Invitrogen | P36934 | Special Packaging |
| Enclosed imaging stage chamber | Warner | RC-30HV | |
| Temperature controller unit | Warner | TC-344B | |
| MetaMorph | Universal Imaging |
References
- Banker, G., Goslin, K. Developments in neuronal cell culture. Nature. 336, 185-186 (1988).
- Xie, Z. Kalirin-7 controls activity-dependent structural and functional plasticity of dendritic spines. Neuron. 56, 640-656 (2007).
- Spear, L. Modeling adolescent development and alcohol use in animals. Alcohol Res Health. 24, 115-123 (2000).
- Srivastava, D. P., Woolfrey, K. M., Liu, F., Brandon, N. J., Penzes, P. Estrogen receptor ss activity modulates synaptic signaling and structure. J Neurosci. 30, 13454-13460 (2010).
- Srivastava, D. P. Rapid enhancement of two-step wiring plasticity by estrogen and NMDA receptor activity. Proc Natl Acad Sci USA. 105, 14650-14655 (2008).
- Woolfrey, K. M. Epac2 induces synapse remodeling and depression and its disease-associated forms alter spines. Nat Neurosci. 12, 1275-1284 (2009).
- Allison, D. W., Gelfand, V. I., Spector, I., Craig, A. M. Role of actin in anchoring postsynaptic receptors in cultured hippocampal neurons: differential attachment of NMDA versus AMPA receptors. J Neurosci. 18, 2423-2436 (1998).
- Harms, K. J., Tovar, K. R., Craig, A. M. Synapse-specific regulation of AMPA receptor subunit composition by activity. J Neurosci. 25, 6379-6388 (2005).
- Xie, Z., Huganir, R. L., Penzes, P. Activity-dependent dendritic spine structural plasticity is regulated by small GTPase Rap1 and its target AF-6. Neuron. 48, 605-618 (2005).
- Jones, K. A. Rapid modulation of spine morphology by the 5-HT2A serotonin receptor through kalirin-7 signaling. Proc Natl Acad Sci USA. 106, 19575-19580 (1957).
- Dunaevsky, A., Tashiro, A., Majewska, A., Mason, C., Yuste, R. Developmental regulation of spine motility in the mammalian central nervous system. Proc Natl Acad Sci USA. 96, 13438-13443 (1999).
- Lichtman, J. W., Conchello, J. A. Fluorescence microscopy. Nat Methods. 2, 910-919 (2005).
- Svoboda, K. Do spines and dendrites distribute dye evenly. Trends Neurosci. 27, 445-446 (2004).
