LETTER-TO-THE-EDITOR

Nitric oxide NMDA signalling in neuromusculartransmission: a missing link in motor end plate diversityand modulation

ZARKO GROZDANOVIC and REINHART GOSSRAU�

Department of Anatomy, University Clinic Benjamin Franklin, Konigin-Luise-Str. 15, D-14195 Berlin, Germany

Received 4 November 1996

The sequence of events leading to skeletal musclecontraction has been unravelled in considerabledetail. The excitation of the muscle fibre takes placeat the neuromuscular junction (NMJ) and is initiatedby nerve impulses, which invoke the release ofacetylcholine (ACh) from axon terminals followingthe influx of Ca2� across the presynaptic membranethrough voltage-gated Ca2� channels. ACh binds tothe nicotinic ACh receptor, a ligand-gated ionchannel, in the post-synaptic muscle fibre membraneand causes an increase in permeability to Na2� andK� (and to a lesser extent also to Ca2� and Mg2�),which produces a local depolarization (end platepotential) sufficient to elicit an action potential. Theelectrical signal is transmitted by the T-tubularsystem to the muscle fibre interior, where it isconverted to mechanical energy by a processdesignated excitation–contraction coupling. This in-volves the activation of the actin–myosin interactionby Ca2� liberated from the sarcoplasmic reticulumthrough the Ca2� release channels (ryanodine recep-tors), which are coupled to voltage sensors (dihy-dropyridine receptors) in the T-tubular membrane.The process is terminated by the action of acetylcho-linesterase, which hydrolyses ACh in the synapticcleft (Engel, 1994; Grinnell, 1995; Ruegg, 1996).

Recent research in several laboratories has identi-fied additional signals besides the anterogradellydirected ACh=nicotineACh receptor pathway, whichmay contribute to the integrity, development andplasticity of the synaptic transmission across theNMJ. The latest hypothesis to be put forward is the

participation of the N-methyl-D-aspartic acid(NMDA) receptor=nitric oxide (NO) signalling cas-cade in bidirectional nerve–muscle interaction. Atcentral synapses, NO is produced post-synapticallyin response to activation of NMDA receptors byglutamate (Garthwaite, 1991). It has been postulatedthat NO serves primarily as a retrograde messengerimpinging upon presynaptic neurons (Dawson et al.,1992). In the account that follows, we shallsummarize the data generated by different groupsworking in different fields, including our own,which point to the involvement of NO in reciprocalneuromuscular transmission and different types ofNMJ.

The story begins with putting together the piecesof evidence in support of a role for glutamate-mediated mechanisms in the vertebrate NMJ.Meister et al. (1993) reported on the expression ofglutamate transporter mRNA in presumable moto-neurons in the spinal cord of the rabbit by in situhybridization. In addition, they localized glutamatein the cell bodies of neurons in the ventral horn ofthe spinal cord and in the region of the NMJ inhindlimb muscles of the rat by light microscopicimmunohistochemistry. Wærhaug and Ottersen(1993) employed ultrastructural immunohistochem-istry to study the distribution of glutamate in NMJof the extensor digitorum and soleus muscles ofrats. Positive labelling was found in nerve terminals,synaptic cleft and muscle fibre cytoplasm. Finally,Berger et al. (1995) were able to demonstrateimmunoreactivity to the NMDA receptor-subuniton the post-junctional sarcolemma of the ratdiagphragm.

Histochemical Journal 29, 267–269 (1997)

0018–2214 # 1997 Chapman & Hall

�To whom correspondence should be sent.

At about the same time, NO came into play. Wehave shown that the brain-type NO synthase (NOSI), the enzyme that catalyses the formation of NOfollowing activation by Ca2�=calmodulin, was pre-sent at the extrajunctional and, especially, junctionalmuscle fibre sarcolemma (Grozdanovic et al., 1995a,b, 1996; Gossrau & Grozdanovic, 1996). Similarly, anaccumulation of NOS I at the NMJ was noted byBrenman et al. (1995) Kusner & Kaminski, (1996)and Oliver et al. (1996). Brenman et al. (1996)succeeded in demonstrating an association of NOSI with Æ1-syntrophin, a dystrophin-linked protein,mediated by PDZ domains. Similar domains existon the post-synaptic density-95 (PSD-95) protein,which serves to link NMDA receptors to NOS I atcentral synapses (Kornau et al., 1995; Bredt, 1996).

In follow-up experiments, we examined the NOSI-containing NMJ in skeletal muscles (tongue,diaphragm, omohyoideus and extensor digitorumlongus muscles) of mice and rats in more detail.Double labelling of NOS I protein or its associatedNADPH diaphorase activity and acetylcholinester-ase activity revealed that NOS I was present in asubset of NMJ with a peculiar topographic distribu-tion within the muscles investigated (Grozdanovic etal., 1996). Additional data show a co-localizationof NMDA receptor-I immunoreactivity and NOS-associated NADPH diaphorase in a minority ofNMJ (Gossrau & Grozdanovic, 1997).

On the basis of these findings, we suggest theexistence of two populations of NMJ in mammalianskeletal muscles, with and without NOS I (type II

and type I respectively). Furthermore, we propose amodel for the activation of NOS I in type II NMJ,which is dependent upon Ca2� inflow from theextracellular space through the NMDA Ca2� chan-nels (Fig. 1). According to this model, glutamate isreleased from presynaptic motor nerve terminalsand binds to NMDA receptors in the post-synapticsarcolemma leading to Ca2� influx and, as aconsequence, stimulation of NOS I, which isassociated with Æ1-syntrophin on the sarcolemma.NO may have multiple functions in the region oftype II NMJ; it may act as a diffusible retrogrademessenger in the reciprocal neuromuscular commu-nication, which may influence the transmissioncharacteristics (e.g. release of transmitter from motornerve terminals, receptor sensitivity), NMJ plasticityand development of the NMJ or modulate phos-phorylation of proteins in the junctional sarcolem-ma, as has been shown for the post-synaptic densityin the rat cerebral cortex (Wu et al., 1996).

Acknowledgements

The authors are grateful to the Ms U. Sauerbier forphotographic work and Ms U. Saykam for thepreparation of the manuscript. The work wassupported by the German Research Foundation(grant BL 259=3-1).

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Acetylcholine Glutamate

Na+ Ca2+

NMDAR

Dystrophin

AChR

CaM NOS I

L-Arginine

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Dystroglycancomplex

Æ1-Syntrophin?

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