Schwann cells and astrocytes induce synapse formation by spinal motor neurons in culture

Abstract

Glia constitute 90% of cells in the human nervous system, but relatively little is known about their functions. We have been focusing on the potential synaptic roles of glia in the CNS. We recently found that astrocytes increase the number of mature, functional synapses on retinal ganglion cells (RGCs) by sevenfold and are required for synaptic maintenance in vitro. These observations raised the question of whether glia similarly enhance synapse formation by other neuron types. Here we have investigated whether highly purified motor neurons isolated from developing rat spinal cords are able to form synapses in the absence of glia or whether glia similarly enhance synapse number. We show that spinal motor neurons (SMNs) form few synapses unless Schwann cells or astrocytes are present. Schwann cells increase the number of functional synapses by ninefold as measured by immunostaining, and increase spontaneous synaptic activity by several hundredfold. Surprisingly, the synapses formed between spinal motor neurons were primarily glutamatergic, as they could be blocked by CNQX. This synapse-promoting activity is not mediated by direct glial–neuronal cell contact but rather is mediated by secreted molecule(s) from the Schwann cells, as we previously found for astrocytes. Interestingly, the synapse-promoting activity from astrocytes and Schwann cells was functionally similar: Schwann cells also promoted synapse formation between retinal ganglion cells, and astrocytes promoted synapse formation between spinal motor neurons. These studies show that both astrocytes and Schwann cells strongly promote synapse formation between spinal motor neurons and demonstrate that glial regulation of synaptogenesis extends to other neuron types.

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.