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Primary cultures of granule neurons from the post-natal rat cerebellum provide an excellent model system for molecular and cell biological studies of neuronal development and function. The cerebellar cortex, with its highly organized structure and few neuronal subtypes, offers a well-characterized neural circuitry. Many fundamental insights into the processes of neuronal apoptosis, migration, and differentiation in the mammalian central nervous system have come from investigating granule neurons in vitro. Granule neurons are the most abundant type of neurons in the brain. In addition to the sheer volume of granule neurons, the homogeneity of the population and the fact that they can be transfected with ease render them ideal for elucidating the molecular basis of neuronal development. This protocol for isolating granule neurons from post-natal rats is relatively straightforward and quick, making use of standard enzymatic and mechanical dissociation methods. In a serum-based medium containing an inhibitor of mitosis, cerebellar granule neurons can be maintained with high purity. Axons and dendrites can be clearly distinguished on the basis of morphology and markers. For even greater versatility, this protocol for culturing granule neurons can be combined with knockout or transgenic technologies, or used in cerebellar slice overlay assays.
Please see Appendices for appropriate handling of materials marked with <!>, and recipes for reagents marked with <R>.
If you are working with two litters, an additional step of trituration is helpful; see Step 11.iv.
Problem: Cerebella are broken upon removal.
Solution: Consider the following:
Problem: Cerebella do not fall apart easily after trypsinization.
Solution: Trypsinize longer, for 15–20 min. If this does not help, make fresh trypsin-DNase aliquots, checking that the concentrations are accurate. When the trypsinization has worked properly, the cerebella should appear softened.
Problem: A viscous pellet is observed after centrifugation.
Solution: The DNase may be spoiled or not concentrated enough. Add 50 µL more of the 2-mg/mL aliquot, and further dissociate the cells.
Problem: Cells clumps are visible during counting.
Solution: Apply more pressure when triturating, and observe the solution carefully for any residual clumps before centrifuging cells.
Problem: There is a low yield of healthy cells and a high rate of cell death.
[Steps 14 and 16]
Solution: There are many causes for this problem. Consider the following:
Problem: Cells clump together after a few days in culture.
Solution: Plating density is probably lower than expected from the count. Be sure not to count the unhealthy cells, which take up trypan blue.
Problem: Cells are not attached, or there are regions of the plate with no cells.
Solution: Consider the following:
Problem: There are too many glia.
Solution: Consider the following:
Cerebellar granule neurons have proven invaluable in uncovering the signaling pathways governing neuronal survival, migration, and differentiation. Studies of neuronal apoptosis have frequently relied on cultures of granule neurons, exploiting their responses to activity and growth factor deprivation as well as oxidative stress. These studies have led to the identification of key neuroprotective molecules including insulin-like growth factor 1, cyclic adenosine monophosphate, phosphatidylinositol-3 kinase, AKT, and myocyte enhancer factor 2 (D’Mello et al. 1993, 1997; Dudek et al. 1997; Mao et al. 1999; Li et al. 2001; Shalizi et al. 2003) and key mediators of neuronal cell death including cyclin-dependent kinase 1, c-Jun N-terminal kinase, and mammalian sterile 20-like kinase (Watson et al. 1998; Harris and Johnson 2001; Konishi et al. 2002; Becker and Bonni 2006; Lehtinen et al. 2006; for review, see Becker and Bonni 2004).
The classic paradigm for studying activity-dependent responses in granule neurons has been the use of membrane-depolarizing concentrations of extracellular potassium chloride (Gallo et al. 1987; Yan et al. 1994). Membrane depolarization activates voltage-sensitive calcium channels, leading to the entry of calcium into neurons and the activation of calcium-dependent signaling molecules, including Ca2+-calmodulin-dependent protein kinases and the phosphatase calcineurin (Sée et al. 2001; Linseman et al. 2003; Wayman et al. 2004; Sato et al. 2005; Suzuki et al. 2005). Calcium-dependent signaling pathways regulate the gene expression profile of cerebellar granule neurons (Kramer et al. 2003; Sato et al. 2005). An interesting example is the developmental switch that occurs in subunit composition for GABAA and NMDA neurotransmitter receptors as granule neurons mature (Watanabe et al. 1992; Farrant et al. 1994; Mathews et al. 1994). Upon hyperpolarization, the α6 subunits of the GABAA receptor and the NR2C subunit of the NMDA receptor are up-regulated (Mellor et al. 1998; Suzuki et al. 2005; for review, see Nakanishi and Okazawa 2006).
Another realm in which granule neurons have occupied center stage is the study of neuronal migration. Granule neurons display a well-known maturation-dependent descent from the external to internal granular layer of the cerebellar cortex. They have been particularly useful in uncovering glial guidance mechanisms for neuronal migration, shedding light on both the cell biology of the migration process and the specific molecular pathways involved. Two crucial regulators of neuronal migration that were identified in studies of cerebellar granule neurons are the adhesion molecule astrotactin and members of the mPar6α polarity complex (Rivas and Hatten 1995; Zheng et al. 1996; Solecki et al. 2004; for review, see Stitt et al. 1991; Solecki et al. 2006).
Most recently, granule neurons have been used to study the cell-intrinsic mechanisms underlying neuronal morphogenesis and connectivity. The transcription factors MEF2 and NeuroD, as well as the ubiquitin ligase Cdh1-APC and its target SnoN, have been identified as key regulators of morphological development in neurons (Gaudillière et al. 2004; Konishi et al. 2004; Shalizi et al. 2006; Stegmüller et al. 2006). For these studies, granule neurons offer the benefit of having a highly stereotypical pattern of polarization with readily distinguishable axons and dendrites. In terms of understanding neuronal connectivity on a broader scale, perhaps the best asset of granule neurons is the relatively simple architecture of the cerebellar cortex, as this may facilitate translating molecular findings about individual cell types into functional consequences for a circuit.
In contrast to the ease of culturing granule neurons, Purkinje neurons—the post-synaptic targets of granule neurons—are notoriously challenging to isolate and maintain in culture. Although recent improvements in culture techniques may increase the survival and differentiation of Purkinje neurons in mixed cerebellar cultures (Furuya et al. 1998; Ito-Ishida et al. 2008), the overall number of Purkinje neurons in such cultures remains quite low. Purifying Purkinje neurons can require Percoll sedimentation and immunopanning technologies (Baptista et al. 1994), which are more expensive and labor-intensive than most neuronal culture protocols. In addition to this difficulty of culturing post-synaptic targets, cerebellar granule neurons develop post-natally, making it difficult to study granule neurons from the many knockout mice that die embryonically or at birth. However, the post-natal development of granule neurons can also be advantageous because the post-natal cerebellum is much easier to isolate than small regions of the embryonic cerebrum.
We apologize to all investigators whose important studies of cerebellar granule neurons could not be cited due to space limitations. We thank members of the Bonni laboratory for refining these culture methods and providing critical readings of the manuscript. Work in the Bonni laboratory is supported by grants from the National Institutes of Health.