Neurotransmitter receptor expression is modulated by activity during both the development and plasticity of mature synapses. N
-aspartic acid (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), kainate, γ-aminobutyric acid A-type (GABAA
) and acetylcholine (ACh) receptor expression are regulated by synaptic activity in a variety of nervous system structures such as the hippocampus, visual cortex, superior colliculus, prefrontal cortex and even in skeletal muscles (Broadie & Bate 1993
; Bessereau et al. 1994
; Catalano et al. 1997
; Kidd & Isaac 1999
; Shi et al. 2000
; Brumwell et al. 2002
At the NMJ, pre- and postsynaptic differentiation appear to be mutually inductive events. Agrin from innervating motor neurons is the trigger for localization of receptors and the associated postsynaptic machinery in muscle cells (Sanes & Lichtman 2001
). Prior to neuronal contact, ACh receptors are distributed largely uniformly over the entire surface of the muscle cell. After neuromuscular contact, ACh receptors cluster rapidly at these sites in the postsynaptic membrane and the density of extrajunctional ACh receptors decreases gradually (Diamond & Miledi 1962
) in a process regulated by muscle cell activity (Lomo & Rosenthal 1972
; Schuetze & Role 1987
; Hall & Sanes 1993
). The role of synaptic transmission in formation of the NMJ was tested in mutant mice lacking choline acetyltransferase activity, the ACh-synthesizing enzyme. The numbers of motor axons, myotubes and Schwann cells were altered. ACh synthesis also seems to have a role in stabilization of nerve–muscle contacts (Misgeld et al. 2002
). In zebrafish embryos, migrating motor axons form en passant synaptic contacts with myotomal muscle before establishing terminal synapses with their final muscle targets. In the twister
mutant, neuromuscular transmission is prolonged at these synapses, and aberrant motor axon trajectories and muscular degeneration ensue (Lefebvre et al. 2004
). In Caenorhabditis elegans
, the postsynaptic muscles can induce sprouting and synaptogenesis in innervating motor neurons (Plunkett et al. 1996
). These studies define clearly the importance of neural activity and cellular interactions in the assembly and refinement of newly formed synapses, but much less is known about the specification of properties of pre- and postsynaptic cells at earlier stages of development.
The vertebrate NMJ has been extensively characterized as cholinergic (Misgeld et al. 2002
). However, several reports have described the expression in skeletal muscle cells of neurotransmitter receptors other than the classical nicotinic receptors. Metabotropic glutamate receptors have been described in the adult frog NMJ (Pinard et al. 2003
), while muscarinic receptors have been found in primary cultures of rat myotubes (Reyes & Jaimovich 1996
). The role of these novel transmitter receptors is unclear, but the investigators suggested a modulation of synaptic events. More recently, Brunelli et al. (2005)
have found AMPA receptors expressed in adult rat skeletal muscle.
We have recently investigated neurotransmitter receptor expression in vertebrate skeletal muscle. Surprisingly, transcripts of several different neurotransmitter receptors in addition to nicotinic cholinergic receptors are present in developing Xenopus
skeletal muscle at an early stage of development, at the onset of muscle innervation (). Moreover, immunolabelling of glutamate and glycine receptor subunits and labelling with tagged muscimol, a GABAA
receptor-specific agonist, reveals that the receptors are present at the protein level in immature skeletal muscle. As maturation and innervation progress, the nicotinic phenotype prevails, while glutamate, GABA and glycine receptor expression is downregulated (a
; Borodinsky & Spitzer 2007
Figure 1 Expression of nAChR, NMDAR, AMPAR, GABAAR and GlyR transcripts in skeletal muscle during normal development. RT-PCR was used for detection of subunit transcripts of five neurotransmitter receptors in muscle, notochord and neural tube at three stages of (more ...)
Figure 2 Expression of nAChR, NMDAR, AMPAR, GABAAR and GlyR protein in skeletal muscle during normal development and after perturbations of Ca spike activity. Whole mounts from 1.3 day (stage 28) embryos and 3 day (stage 40) control (a) and activity-manipulated (more ...)
We then investigated whether this developmental selection of neurotransmitter receptor expression in muscle is activity dependent. Indeed, early neuronal activity regulates neurotransmitter receptor selection in the skeletal muscle. When activity is suppressed, leading to increases in the number of cholinergic and glutamatergic neurons, glutamate receptors remain. In contrast, when activity is enhanced, leading to increases in the number of GABAergic and glycinergic neurons, GABAA and glycine receptors remain, in addition to nicotinic cholinergic receptors (b).
Since synaptic boutons immunopositive for glutamate, GABA or glycine were detected in embryos in which activity has been perturbed, we next determined whether non-cholinergic NMJs were formed upon alterations of neuronal activity. Remarkably, glutamatergic synaptic currents were recorded following suppression of activity, and GABAergic and glycinergic synaptic currents were evident when activity was enhanced (; Borodinsky & Spitzer 2007
). The findings are consistent with observations of the release of glutamate from neonatal mammalian motor neurons (Mentis et al. 2005
; Nishimaru et al. 2005
). Glutamatergic innervation of adult rat skeletal muscle via peripheral nerve grafts (Brunelli et al. 2005
) may result from these activity-dependent processes.
Figure 3 Matching of neurotransmitters and receptors at NMJs in vivo. Whole-cell recordings from muscle cells of the axial musculature of 3 day (stage 40) (a) control, (b) Ca2+ spike-suppressed and (c) Ca2+ spike-enhanced larvae were performed in the presence (more ...)
These results demonstrate that (i) multiple classes of receptors are expressed embryonically, (ii) these classes are pruned following innervation, and (iii) there is a mechanism for clustering the appropriate receptors under presynaptic terminals. The matching of transmitters with their cognate receptors by a process of selection from a pool of receptor classes may seem inefficient. However, it could facilitate rapid establishment of connections and has a parallel with other biological processes—most notably antibody selection in the immune system.
We emphasize that there are limits to what activity can achieve. It does not act on a tabula rasa but is constrained by genetically determined neuronal identity. We model this process as the sequential channelling of cell fates to progressively more restricted phenotypes by differential gene expression, and envisage that different populations of neurons finally have the capability to express one or more of a series of neurotransmitters. Similarly, we propose that populations of neurons, muscles and glands express a substantial number of different transmitter receptors. Patterns of electrical activity in presynaptic neurons then determine both the final outcome of neurotransmitter expression and the match between presynaptic neurotransmitter and postsynaptic receptors ().
Figure 4 Model illustrating the process of transmitter–receptor matching at the NMJ. Prior to innervation, muscle cells express multiple classes of receptors. During normal development, motor neurons express ACh and AChR that persist on the muscle fibres. (more ...)