Protocol for Evaluating Neuronal Polarity in Murine Models

Frontiers in Cell and Developmental Biology

Axon-dendrite formation is a crucial milestone in the life history of neurons. During this process, historically referred as “the establishment of polarity,” newborn neurons undergo biochemical, morphological and functional transformations to generate the axonal and dendritic domains, which are the basis of neuronal wiring and connectivity. Since the implementation of primary cultures of rat hippocampal neurons by Gary Banker and Max Cowan in 1977, the community of neurobiologists has made significant achievements in decoding signals that trigger axo-dendritic specification. External and internal cues able to switch on/off signaling pathways controlling gene expression, protein stability, the assembly of the polarity complex (i.e., PAR3-PAR6-aPKC), cytoskeleton remodeling and vesicle trafficking contribute to shape the morphology of neurons. Currently, the culture of hippocampal neurons coexists with alternative model systems to study neuronal polarization in several species, from s...

Journal of Cell Biology, 1989

In culture, hippocampal neurons develop a polarized form, with a single axon and several dendrites. Transecting the axons of hippocampal neurons early in development can cause an alteration of polarity; a process that would have become a dendrite instead becomes the axon (Dotti, C. G., and G. A. Banker. 1987. Nature (Lond.). 330:254-256). To investigate this phenomenon more systematically, we transected axons at varying lengths. The greater the distance of the transection from the soma, the greater the probability for regrowth of the original axon. However, it was not the absolute length of the axonal stump that determined the response to transection, but rather its length relative to the lengths of the cell's other processes. If one process was greater than 10 microns longer than the others, it invariably became the axon regardless of its identity before transection. Conversely, when a cell's processes were nearly equal in length, it was impossible to predict which would be...

Developmental Biology, 1989

By the end of the first week in culture, hippocampal neurons have established a single axon and several dendrites. These 2 classes of processes differ in their morphology, in their molecular composition,

Journal of Visualized Experiments, 2012

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5 . To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8 . Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.

Journal of Neuroscience Methods, 1998

The Journal of Neuroscience, 2018

In 1988, Carlos Dotti, Chris Sullivan, and I published a paper on the establishment of polarity by hippocampal neurons in culture, which continues to be frequently cited 30 years later (Dotti et al., 1988). By following individual neurons from the time of plating until they had formed well developed axonal and dendritic arbors, we identified the five stages of development that lead to the mature expression of neuronal polarity. We were surprised to find that, before axon formation, the cells pass through a multipolar phase, in which several, apparently identical short neurites undergo periods of extension and retraction. Then one of these neurites begins a period of prolonged growth, becoming the definitive axon; the remaining neurites subsequently become dendrites. This observation suggested that any of the initial neurites were capable of becoming axons, a hypothesis confirmed by later work. In this Progressions article, I will try to recall the circumstances that led to this work...

Current Biology, 2008

Current Opinion in Cell Biology, 2012

In a biological sense, polarity refers to the extremity of the main axis of an organelle, cell, or organism. In neurons, morphological polarity begins with the appearance of the first neurite from the cell body. In multipolar neurons, a second phase of polarization occurs when a single neurite initiates a phase of rapid growth to become the neuron's axon, while the others later differentiate as dendrites. Finally, during a third phase, axons and dendrites develop an elaborate architecture, acquiring special morphological and molecular features that commit them to their final identities. Mechanistically, each phase must be preceded by spatial restriction of growth activity. We will review recent work on the mechanisms underlying the polarized growth of neurons.

2012

Neurons are highly polarized cells and typically develop a single axon and several highly branched dendrites. The establishment of a polarized morphology and the specialization of axonal and dendritic compartments are essential steps in the differentiation of neurons . To study neuronal polarization in vitro, cultures of hippocampal neurons have been extensively used. As neurons develop, they form several neurites, but at some point they begin to polarize so that one neurite becomes an axon whereas the remaining neurites become dendrites (Arimura and Kaibuchi 2007). This asymmetric neurite formation is regulated by molecules that control cytoskeletal rearrangement and protein trafficking.

Current Opinion in Neurobiology, 1997

The mechanisms that permit neurons to establish axons and dendrites involve an interplay between a cell's genetic program and signals in its environment. Recent experiments have identified some of the important extracellular molecules that regulate dendritic development and have furthered our understanding of the endogenous cell biological mechanisms that underlie protein sorting. Some of the signaling pathways that allow extracellular cues to regulate neuronal morphogenesis are also being elucidated.