Neurotransmitters as early signals for central nervous system development.

By Nguyen L., Mangin J.-M., Belachew S., Rogister B., Moonen G. and Rigo J.M. (in collaboration with P. Legendre, INSERM, Paris)

(Research conducted in the: "Centre de Recherches en Neurobiologie Cellulaire et Moléculaire, Université de Liège, Faculté de Médecine) 

During brain ontogenesis, the temporal and spatial generation of different types of neuronal and glial cells from precursors occurs as a sequence of successive progenitor stages whose proliferation, survival and cell fate choice are controlled by environmental and cellular regulatory molecules. Neurotransmitters belong to the chemical microenvironment of neural cells, even at the earliest stages of brain development. It is now well established that specific neurotransmitter receptors are present on progenitor cells of the developing nervous system and could play, during development, a role that has remained unsuspected until recently.

Gamma-aminobutyric acid (GABA) and its type A receptor (GABAAR) are present in the immature central nervous system (CNS) and may function as growth-regulatory signals during the development of embryonic neural precursor cells. Based on their isopycnic properties in a buoyant density gradient, we developed an isolation procedure that allowed us to purify proliferative neural precursor cells from early postnatal rat striatum, which expressed the polysialylated form of the neural cell adhesion molecule (PSA-NCAM). These postnatal striatal PSA-NCAM+ cells were shown to proliferate in the presence of epidermal growth factor (EGF) and formed spheres that generated preferentially neurons in vitro. We demonstrated that PSA-NCAM+ neuronal precursors from postnatal striatum expressed GABAAR subunits in vitro and in situ (Nguyen et al., 2003). GABA elicited chloride currents in PSA-NCAM+ cells by activation of functional GABAAR that displayed a typical pharmacological profile. GABAAR activation in PSA-NCAM+ cells triggered a complex intracellular signaling combining a tonic inhibition of the mitogen-activated protein kinase cascade and an increase of intracellular calcium concentration by opening of voltage-gated calcium channels. We observed that the activation of GABAAR in PSA-NCAM+ neuronal precursors from postnatal striatum inhibited cell cycle progression both in neurospheres and in organotypic slices. Furthermore, postnatal PSA-NCAM+ striatal cells synthesized and released GABA, thus creating an autocrine/paracrine mechanism that controls their proliferation. We showed that EGF modulated this autocrine/paracrine loop by decreasing GABA production in PSA-NCAM+ cells. This demonstration of GABA synthesis and GABAAR function in striatal PSA-NCAM+ cells may shed a new light in the understanding of key extrinsic cues that regulate the developmental potential of postnatal neuronal precursors in the CNS.

We next reported by immunocytochemical analysis that ionotropic glycine receptors (GlyRs) are expressed in PSA-NCAM+ neuronal precursors from postnatal striatum, both in vitro and in situ (Nguyen et al., 2004). To ascertain whether these GlyRs were functional in vitro, whole-cell patch-clamp recordings of neurogenic PSA-NCAM+ progenitors demonstrated that glycine triggered inward strychnine-sensitive currents in the majority of these cells. Moreover, we found that GlyRs expressed by these neurogenic progenitors display intermediate electrophysiological characteristics between those of glycine receptors expressed by neural stem cells and by mature interneurons from the rat striatum. These data further support the view that functional GlyRs, as other neurotransmitter receptors, are expressed by a wide range of immature neural cells: multipotent progenitors (Nguyen et al., 2002), oligodendrogenic (Belachew et al., 1998) and neurogenic progenitors derived from the postnatal brain (Nguyen et al., 2004). Immature nerve cells are known, at least in the spinal cord, to express α2 homomeric GlyRs, the properties of which are relatively unknown compared to those of the adult synaptic form of the GlyR (mainly α1β heteromeres). In a recent work (Mangin et al., 2003), we have recorded the kinetics properties of GlyRs at the single-channel level in real-time by means of the patch-clamp technique in the outside-out configuration coupled with an ultra-fast flow application system (< 100 µs). Recordings were performed on CHO cells stably transfected with the α2 GlyR subunit. We show that the onset, the relaxation and the desensitization of α2 homomeric GlyR-mediated currents are slower by one or two order of magnitude compared to synaptic mature GlyRs and to other ligand-gated ionotropic channels involved in fast synaptic transmission. First latency analysis performed on single GlyR channels revealed that their slow activation time-course was due to delayed openings. When synaptic release of glycine was mimicked (1 mM glycine; 1 ms pulse duration), the opening probability of α2 homomeric GlyRs was low (Po ≈ 0.1) when compared to mature synaptic GlyRs (Po = 0.9). This low Po is likely to be a direct consequence of the relatively slow activation kinetics of α2 homomeric GlyRs when compared to the activation kinetics of mature α1/β GlyRs. Such slow kinetics suggest that embryonic α2 homomeric GlyRs cannot be activated by fast neurotransmitter release at mature synapses but rather could be better suited for a non-synaptic paracrine-like release of agonist, which is known to occur in the embryo. These functional properties of embryonic GlyRs will be further studied in oligodendrogenic and neurogenic progenitor cells, both in vitro and in vivo. The potential implication of the embryonic GlyR in the control of the survival, the proliferation and the differentiation of these progenitors will also be investigated.


Reference List

1. Belachew S, Rogister B, Rigo J-M, Malgrange B, Mazy-Servais C, Xhauflaire G, Coucke P, Moonen G (1998) Cultured oligodendrocyte progenitors derived from cerebral cortex express a glycine receptor which is pharmacologically distinct from the neuronal isoform. Eur J Neurosci 10: 3556-3564.

2. Mangin JM, Baloul M, Prado DC, Rogister B, Rigo JM, Legendre P (2003) Kinetic properties of the alpha2 homo-oligomeric glycine receptor impairs a proper synaptic functioning. J Physiol 553: 369-386.

3. Nguyen L, Malgrange B, Belachew S, Rogister B, Rocher V, Moonen G, Rigo J-M (2002) Functional glycine receptors are expressed by postnatal nestin positive neural stem/progenitor cells. Eur J Neurosci 15: 1299-1305.

4. Nguyen L, Malgrange B, breuskin I, Belachew S, Bettendorf L, Moonen G, Rigo J-M (2003) Autocrine/paracrine activation of the GABAA receptor inhibits the proliferation of neurogenic polysialylated neural cell adhesion molecule-positive (PSA-NCAM+) precursor cells from postnatal striatum. J Neurosci 23: 3278-3294.

5. Nguyen L, Malgrange B, breuskin I, Lallemend F, Hans G, Moonen G, Belachew S, Rigo JM (2004) Striatal PSA-NCAM(+) precursor cells from the newborn rat express functional glycine receptors. nr 15: 583-587.