Schwann cells and astrocytes induce synapse formation by spinal motor neurons in culture
Introduction
Two glial cell types, astrocytes and Schwann cells, ensheath CNS and PNS synapses, respectively. Synaptic glia clear extracellular ions and neurotransmitters from the synaptic cleft and recent studies suggest that astrocytes actively regulate synapse formation, function, and stability Araque et al., 1998, Araque et al., 1999, Bergles and Jahr, 1998, Fields and Stevens-Graham, 2002, Nagler et al., 2001, Pfrieger and Barres, 1997, Ullian et al., 2001, Ventura and Harris, 1999. For example, astrocytes in culture sense and respond to neuronal activity by increasing their internal Ca2+ concentration, which in turn triggers them to release their own chemical transmitters that regulate neuronal activity (Araque et al., 1999). Perisynaptic Schwann cells also detect synaptic activity by increasing intracellular Ca2+ and respond to this activity by regulating neurotransmitter release Reist and Smith, 1992, Rochon et al., 2001. These studies strongly suggest that these two perisynaptic glial cells, astrocytes and Schwann cells, are intimately involved in the process of synaptic function.
In order to address the functions of perisynaptic glial cells, we have been studying the interactions of highly purified populations of neurons and glia in culture. We recently found that purified retinal ganglion cells (RGCs) form few synapses in vitro in the absence of astrocytes. When astrocytes or astrocyte-conditioned media are added to the RGCs, however, the number of synapses that form significantly increases (Ullian et al., 2001) helping to account for earlier studies showing that RGCs cultured in the presence of astrocyte-conditioned medium had about 10–100 times higher synaptic activity (Pfrieger and Barres, 1997). These studies provided evidence for an active role for astrocytes in promoting CNS synaptogenesis and raised the question of whether glial cells similarly promote synaptogenesis by other types of neurons.
In this study, we have addressed the possible role of glial cells in promoting synaptogenesis by spinal motor neurons. By culturing the spinal motor neurons in serum-free medium with high levels of neurotrophic support that ensures that the majority of neurons survive in the total absence of glial cells, we are able to quantitatively investigate the possible requirement for glial cells to promote synaptogenesis. We find that highly purified spinal motor neurons are unable to form synapses upon each other unless Schwann cells or medium conditioned by Schwann cells are present. Moreover, the synaptogenic effects of astrocytes and Schwann cells are interchangeable, each strongly promoting synaptogenesis in cultures of purified retinal ganglion cells or spinal motor neurons. These studies suggest that the requirement for glial cells to promote synaptogenesis may be a more general one, regulating synapse formation by many different types of neurons, and further show that in addition to astrocytes, Schwann cells strongly enhance synaptogenesis.
Section snippets
Effects of Schwann cells on spinal motor neuron synaptogenesis
We first investigated the ability of Schwann cells to enhance synaptogenesis by spinal motor neurons (SMNs). We chose to study their interaction for two reasons. First, because Schwann cells normally ensheath SMN terminals at the neuromuscular synaptic junction, and second, because it is possible to highly purify spinal motor neurons away from glia and other cell types and maintain the survival of the majority of purified SMNs in a defined serum-free medium Camu and Henderson, 1992, Hanson et
Spinal motor neurons in culture form few synapses in the absence of glia
By studying the interactions of highly purified populations of rat spinal motor neurons and Schwann cells in vitro, we have shown that few functional synapses are formed by purified SMNs alone, but that Schwann cells profoundly enhance the number of synapses by nearly 10-fold. Previous studies have found that astrocytes greatly enhance the number of synapses formed by purified RGCs in culture. Similarly to these previous studies, because we cultured the purified SMNs in serum-free medium with
Experimental methods
Step-by-step protocols for all procedures available upon request to B. Barres at [email protected].
Acknowledgements
This work was supported by NIDA (R01 DA15043) (B.A.B.), a Zaffaroni fellowship (E.M.U.), and NINDS (K08 NS02145) (B.T.H.), and an HHMI undergraduate fellowship (A.W.). We thank Regeneron for generously providing BDNF and CNTF.
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These authors contributed equally to this paper.