Abstract
In neonatal mice motoneurons excite Renshaw cells by releasing both acetylcholine (ACh) and glutamate. These two neurotransmitters activate two types of nicotinic receptors (nAChRs) (the homomeric α7 receptors and the heteromeric α*ß* receptors) as well as the two types of glutamate receptors (GluRs) (AMPARs and NMDARs). Using paired recordings, we confirm that a single motoneuron can release both transmitters on a single post-synaptic Renshaw cell. We then show that co-transmission is preserved in adult animals. Kinetic analysis of miniature EPSCs revealed quantal release of mixed events associating AMPARs and NMDARs, as well as α7 and α*ß* nAChRs, but no evidence was found for mEPSCs associating nAChRs with GluRs. Bayesian Quantal Analysis (BQA) of evoked EPSCs showed that the number of functional contacts on a single Renshaw cell is more than halved when the nicotinic receptors are blocked, confirming that the two neurotransmitters systems are segregated. Our observations can be explained if ACh and glutamate are released from common vesicles onto spatially segregated post-synaptic receptors clusters, but a pre-synaptic segregation of cholinergic and glutamatergic release sites is also possible.
Highlights
The early view that chemical synaptic transmission is mediated by a single transmitter activating a single-receptor type has evolved since the observation that at some synapses a transmitter activates multiple classes of post-synaptic receptors, and that a given neuron can release multiple transmitters[1]
In three pairs of connected MNs and Renshaw cell (RC) we demonstrated co-transmission using the rectification properties of the two nicotinic currents, blocked when the cell is held at positive potentials, and those of the NMDARs, that are blocked by external Mg2+ ions when the RC is hyperpolarized[6]
In order to circumvent this difficulty, we examined if “asynchronous EPSCs” could be evoked by stimulation of the MN axons. aEPSCs have been shown in other systems to correspond to “uniquantal” events at synaptic terminals recently invaded by an action potential”[23], and have been used to isolate quantal events associated with a defined set of stimulated synapses[24]
Summary
The early view that chemical synaptic transmission is mediated by a single transmitter activating a single-receptor type has evolved since the observation that at some synapses a transmitter activates multiple classes of post-synaptic receptors, and that a given neuron can release multiple transmitters[1]. If the two receptors are co-localized and have different kinetics, spontaneous quantal release will give rise to miniature post synaptic currents (mPSCs) with mixed kinetics, a feature that was used to demonstrate co-localization of AMPARs and NMDARs in cultured neurons[7,8,9,10,11,12] and of nicotinic receptors containing α3 subunits and α7 nAChRs in the chicken ciliary ganglion[13, 14]. We exploited the differences in kinetics among the nAChRs and GluRs and detected both mixed glutamatergic and mixed cholinergic mEPSCs, but never mEPSCs associating a nAChR and a GluR This segregation was confirmed by quantal analysis of evoked EPSCs which showed a specific reduction in the number of functional release sites following pharmacological blockade of nAChRs
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