Stoichiometry of the Heteromeric Nicotinic Receptors of the Renshaw Cell.

Neuronal nicotinic acetylcholine receptors (nAChRs) are pentamers built from a variety of subunits. Some are homomeric assemblies of α subunits, others heteromeric assemblies of α and ß subunits which can adopt two stoichiometries (2α:3ß or 3α:2ß). There is evidence for the presence of heteromeric nAChRs with the two stoichiometries in the CNS, but it has not yet been possible to identify them at a given synapse. The 2α:3ß receptors are highly sensitive to agonists, whereas the 3α:2ß stoichiometric variants, initially described as low sensitivity receptors, are indeed activated by low and high concentrations of ACh. We have taken advantage of the discovery that two compounds (NS9283 and Zn) potentiate selectively the 3α:2ß nAChRs to establish (in mice of either sex) the presence of these variants at the motoneuron-Renshaw cell (MN-RC) synapse. NS9283 prolonged the decay of the two-component EPSC mediated by heteromeric nAChRs. NS9283 and Zn also prolonged spontaneous EPSCs involving heteromeric nAChRs, and one could rule out prolongations resulting from AChE inhibition by NS9283. These results establish the presence of 3α:2ß nAChRs at the MN-RC synapse. At the functional level, we had previously explained the duality of the EPSC by assuming that high ACh concentrations in the synaptic cleft account for the fast component and that spillover of ACh accounts for the slow component. The dual ACh sensitivity of 3α:2ß nAChRs now allows to attribute to these receptors both components of the EPSC.SIGNIFICANCE STATEMENT Heteromeric nicotinic receptors assemble α and ß subunits in pentameric structures, which can adopt two stoichiometries: 3α:2ß or 2α:3ß. Both stoichiometric variants are present in the CNS, but they have never been located and characterized functionally at the level of an identified synapse. Our data indicate that 3α:2ß receptors are present at the spinal cord synapses between motoneurons and Renshaw cells, where their dual mode of activation (by high concentrations of ACh for synaptic receptors, by low concentrations of ACh for extrasynaptic receptors) likely accounts for the biphasic character of the synaptic current. More generally, 3α:2ß nicotinic receptors appear unique by their capacity to operate both in the cleft of classical synapses and at extrasynaptic locations.

Segregation of glutamatergic and cholinergic transmission at the mixed motoneuron Renshaw cell synapse.

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.

High affinity and low affinity heteromeric nicotinic acetylcholine receptors at central synapses.

Most neuronal heteromeric nicotinic receptors seem able to adopt two different stochiometries depending on the ratio of α and ß subunits. In recombinant receptors these two stoichiometries have been associated with different affinities to ACh, but it is not known which stoichiometry is present at nicotinic synapses in the nervous system. One possible clue to this identification is the speed of decay of the synaptic currents. In many ionotropic receptors this speed has been linked to the dissociation rate of the transmitter, which is itself related to its affinity. On this basis we propose that, at the synapse between motoneuron and Renshaw cells, the heteromeric nicotinic receptors are mostly low affinity receptors and suggest that, in contrast, the very slow decay of some synaptic currents recorded in other parts of the brain signs the presence of high affinity receptors rather than volume transmission.

Subunit composition and kinetics of the Renshaw cell heteromeric nicotinic receptors.

In Renshaw cells (RCs) of newborn mice, activation of motoneurons elicits a four-component synaptic current (EPSC) mediated by two glutamate receptors and two nicotinic receptors (nAChRs). We have analyzed the nicotinic component of the EPSC which is blocked by dihydro-beta-erythroidine (DHßE) with the dual objective of identifying the nAChR subunits involved and of understanding the kinetics of the response. The sensitivity to DHßE of the peak of the EPSC was differentially affected by genetic deletion of three specific nAChR subunits: α2, ß2 and ß4. The comparison of these effects with published findings on recombinant receptors suggests that, in WT mice, two heteromeric assemblies, α4ß2 and α2ß4, coexist in variable proportions in a given RC. Some results seem to require, however, the involvement of an additional subunit. The effects of DHßE on the decay of the EPSCs were compared in WT mice and in PRiMA(-/-) mice, in which the decay is prolonged by the absence of central acetylcholinesterase. In PRiMA(-/-) mice DHßE shortened the decay of the EPSC. In WT mice it did not alter the decay but reduced the amplitude of both components of the EPSC. The results can be interpreted by assuming that the nAChRs exist in two stoichiometries, subsynaptic "low sensitivity" nAChRs and extrasynaptic "high sensitivity" nAChRs activated by spillover.

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Near-complete adaptation of the PRiMA knockout to the lack of central acetylcholinesterase.

Acetylcholinesterase (AChE) rapidly hydrolyzes acetylcholine. At the neuromuscular junction, AChE is mainly anchored in the extracellular matrix by the collagen Q, whereas in the brain, AChE is tethered by the proline-rich membrane anchor (PRiMA). The AChE-deficient mice, in which AChE has been deleted from all tissues, have severe handicaps. Surprisingly, PRiMA KO mice in which AChE is mostly eliminated from the brain show very few deficits. We now report that most of the changes observed in the brain of AChE-deficient mice, and in particular the high levels of ambient extracellular acetylcholine and the massive decrease of muscarinic receptors, are also observed in the brain of PRiMA KO. However, the two groups of mutants differ in their responses to AChE inhibitors. Since PRiMA-KO mice and AChE-deficient mice have similar low AChE concentrations in the brain but differ in the AChE content of the peripheral nervous system, these results suggest that peripheral nervous system AChE is a major target of AChE inhibitors, and that its absence in AChE- deficient mice is the main cause of the slow development and vulnerability of these mice. At the level of the brain, the adaptation to the absence of AChE is nearly complete.

Mechanisms shaping the slow nicotinic synaptic current at the motoneuron-renshaw cell synapse.

In spinal cord slices from newborn mice we have analyzed the kinetics of the EPSCs mediated by heteromeric nicotinic receptors at the motoneuron-Renshaw cell (MN-RC) synapse. The miniature EPSCs decay with a time constant of 13.0 ± 1.1 ms whereas the decay of the evoked EPSCs (eEPSCs) is biphasic, with time constants of 15.6 ± 0.8 and 124.8 ± 9.0 ms. The slow component becomes prominent during a repetitive stimulation, but its time constant is unchanged. It is selectively reduced by the addition of acetylcholinesterase (AChE), and thus appears to involve ACh spillover. The constancy of the slow time constant during a train is best explained by a local spillover activating high-affinity receptors. In many cells a fraction of the eEPSC originates in neighboring RCs and is transmitted by the low-pass filter of the gap junctions. The component transmitted electrically can be eliminated by meclofenamic acid, a blocker of gap junctions. The local spillover produced by a repetitive stimulation was compared with the long-range spillover produced by inactivation of AChE. The pharmacological inactivation of AChE by neostigmine caused the appearance of an ultra-slow (second range) decay component in eEPSCs and also a continuous inward current interpreted as resulting from a continuous ACh presence. In animals lacking functional AChE in the CNS (PRiMA(-/-) mice) the EPSCs resembled those observed in neostigmine but the steady inward current was much smaller, suggesting an adaptation to the absence of AChE.

Four excitatory postsynaptic ionotropic receptors coactivated at the motoneuron-Renshaw cell synapse.

Renshaw cells (RCs) are spinal interneurons excited by collaterals of the axons of motoneurons (MNs). They respond to a single motoneuronal volley by a surprisingly long (tens of milliseconds) train of action potentials. We have analyzed this synaptic response in spinal cord slices of neonatal mice in light of recent observations suggesting that the MN axons release both acetylcholine and glutamate. We found that the RC synaptic current involves four components of similar amplitudes mediated by two nicotinic receptors (nAChRs, tentatively identified as alpha(7) homomers and alpha(4)beta(2) heteromers) and two glutamate receptors (AMPARs and NMDARs). The decay time constants of the four components cover a wide range: from 3.6 +/- 2.2 ms (alpha(7) nAChRs) to 54.6 +/- 19.5 ms (NMDARs, at -45 mV). The RC discharge can be separated into an initial doublet of high-frequency action potentials followed by later spikes with a variable latency and longer interspike intervals. The initial doublet involves the four ionotropic receptors as well as endogenous voltage-dependent conductances. The late discharge depends on NMDARs, but these receptors must be primed by the initial depolarization. The activation of the NMDARs is prolonged by the fact that their slow deactivation is further slowed by depolarization. The formation of the initial doublet is favored by hyperpolarization, whereas the late discharge is favored by depolarization. This suggests that in physiological conditions the pattern of discharge of the RC in response to a MN input may alternate between a phasic and a tonic response.

Repetitive firing of rat cerebellar parallel fibres after a single stimulation.

The excitatory postsynaptic currents (EPSCs) evoked in Purkinje cells (PCs) by stimulating parallel fibres (PFs) usually show a single peak, but EPSCs with multiple peaks (polyphasic EPSCs) can be observed in slices from animals older than 15 days. The EPSCs remain polyphasic when the postsynaptic current is reduced (either by reducing the intensity of the PF stimulation or by adding AMPA receptor antagonists) and when the PC membrane potential is made positive. Thus the late peaks are not due to postsynaptic active currents generated in the imperfectly clamped PC, and must arise from repetitive action potentials in the PF. Extracellular recordings from granule cell (GC) somata showed that a single PF stimulation can elicit a doublet or a train of action potentials. Both the late action potentials recorded in the GCs and the late peaks of the polyphasic EPSCs recorded in the PCs were reduced or abolished by paired-pulse stimulation of the PF or by bath application of the GABA(A) agonist muscimol. The late action potentials in the GCs were also suppressed by local application of muscimol around the cell body. We propose that after a single stimulation of a PF, the antidromic invasion of the ascending axon and the granule cell can trigger a doublet or a burst of action potentials which back-propagate into the PF (except for the first, which finds the PF still in its refractory period). The repetitive activation of the PF by a single stimulation could play a role in the induction of long-term depression.

Involvement of presynaptic N-methyl-D-aspartate receptors in cerebellar long-term depression.

At the cerebellar synapses between parallel fibers (PFs) and Purkinje cells (PCs), long-term depression (LTD) of the excitatory synaptic current has been assumed to be independent of the N-methyl-D-aspartate (NMDA) receptor activation because PCs lack NMDA receptors. However, we now report that LTD is suppressed by NMDA receptor antagonists that act on presynaptic NMDA receptors of the PFs. This effect is still observed when the input is restricted to a single fiber. Therefore, LTD does not require the spatial integration of multiple inputs. In contrast, it involves a temporal integration, since reliable LTD induction requires the PFs to fire two action potentials in close succession. This implies that LTD will selectively depress the response to a burst of presynaptic action potentials.