Mammalian motor neurons corelease glutamate and acetylcholine at central synapses

Abstract

Motor neurons (MNs) are the principal neurons in the mammalian spinal cord whose activities cause muscles to contract. In addition to their peripheral axons, MNs have central collaterals that contact inhibitory Renshaw cells and other MNs. Since its original discovery >60 years ago, it has been a general notion that acetylcholine is the only transmitter released from MN synapses both peripherally and centrally. Here, we show, using a multidisciplinary approach, that mammalian spinal MNs, in addition to acetylcholine, corelease glutamate to excite Renshaw cells and other MNs but not to excite muscles. Our study demonstrates that glutamate can be released as a functional neurotransmitter from mammalian MNs.synaptic transmission ͉ spinal cord M otor neurons (MNs) are the output neurons from the central nervous system. Their activity directly leads to muscle contraction. By the 1940s, it was generally accepted that MNs release acetylcholine (ACh) at the neuromuscular junction (1). Shortly thereafter, it was shown that ACh also is released from MNs' central axonal branches contacting Renshaw cells (RCs) (2). As was found at the neuromuscular junction, this transmission was shown to be nicotinic (3, 4). The collaterals contacting other MNs (5) are also thought to be mediated by ACh, although this has not been shown directly (6). Since these initial discoveries and after many later investigations, it has been a general dogma that mammalian MNs contain and release one neurotransmitter, ACh, both centrally and peripherally. It has been suggested recently, based on anatomical data, that MNs might contain glutamate as a neurotransmitter (7,8). There has been, however, no direct electrophysiological evidence to support this. Here, we examine this question directly by investigating the transmission in central and peripheral MN synapses (Fig. 1a). Materials and MethodsRecordings from RCs and MNs. All procedures followed Swedish federal guidelines for animal care. Postnatal heterozygote glutamic acid decarboxylase (GAD) 67-GFP mice [postnatal day (P) 0 to P4] were anaesthetized with isoflurane and eviscerated, and spinal cords were removed with ventral laminectomy, as described in ref. 9. The spinal cord was placed in a recording chamber perfused with oxygenated Ringer's solution (128 mM NaCl͞4.69 mM KCl͞25 mM NaHCO 3 ͞1.18 mM KH 2 PO 4 ͞1.25 mM MgSO 4 ͞2.5 mM CaCl 2 ͞22 mM glucose aerated with 5% CO 2 in O 2 ) at room temperature. Whole-cell tight-seal recording of RCs and MNs were performed with patch electrodes pulled from thick-walled borosilicate glass (o.d. of 1.5 mm, i.d. of 1.0 mm; Harvard Instruments) to a final resistance of 5-8 M⍀. The electrode tips were filled with 138 mM K-gluconate, 10 mM Hepes, 0.0001 mM CaCl 2 , 5 mM ATP-Mg, and 0.3 mM GTP-Li. After filling of the tip, the electrodes were back-filled with the same solution, and to label the recorded cell, Alexa Fluor dye (0.15-0.20%; Molecular Probes) or neurobiotin (1-2%) was diluted into the electrode solution. Cells were filled during recording. Signals wer...

Activity-dependent neurotransmitter-receptor matching at the neuromuscular junction

“…The results are in agreement with findings of glutamatergic transmission at adult vertebrate NMJs (34) and with the release of glutamate from neonatal mammalian motorneurons (35,36). Glutamatergic-cholinergic cotransmission at the vertebrate NMJ may contribute to apparently normal behavioral and physiological development after chronic blockade of embryonic electrical activity (37,38).…”

Glutamatergic reinnervation through peripheral nerve graft dictates assembly of glutamatergic synapses at rat skeletal muscle

“…Although VGluT-1͞2 transcripts have been detected in rat spinal motoneurons (28,29), no VGluT-1͞2 proteins have been found to colocalize with VAChT (28,30) or ChAT (31). Recent studies demonstrating VGluT-2 immunostaining in motoneuron collateral axons projecting to layer VII of the spinal cord (28,32) confirmed the lack of VGluT-2 and glutamatergic transmission in the cholinergic projections to the NMJ. Furthermore, studies on VGluT-3 expression at the skeletal muscle endplates gave contradictory results (33,34).…”

Spinal cholinergic interneurons regulate the excitability of motoneurons during locomotion

“…Although there are other cholinergic inputs to spinal MNs besides C boutons (6,31), several lines of evidence suggest that C boutons are responsible for the increased MN excitability during locomotor-related activity. First, being on or close to MN somata, C boutons are well positioned to act as modulators of the intrinsic properties, such as the AHP, that regulate neuronal output.…”