Cholinergic partition cells and lamina x neurons induce a muscarinic-dependent short-term potentiation of commissural glutamatergic inputs in lumbar motoneurons

Abstract

Acetylcholine and the activation of muscarinic receptors influence the activity of neural networks generating locomotor behavior in the mammalian spinal cord. Using electrical stimulations of the ventral commissure, we show that commissural muscarinic (CM) depolarizations could be induced in lumbar motoneurons. We provide a detailed electrophysiological characterization of the muscarinic receptors and the membrane conductance involved in these responses. Activation of the CM terminals, originating from lamina X neurons and partition cells, induced a pathway-specific short-term potentiation (STP) of commissural glutamatergic inputs in motoneurons. This STP is occluded in the presence of the muscarinic antagonist atropine. During fictive locomotion, the activation of the commissural pathways transiently enhanced the motor output in a muscarinic-dependent manner. This study describes for the first time a novel regulatory mechanism of synaptic strength in spinal locomotor networks. Such cellular mechanisms would endow the locomotor central pattern generators with adaptive processes needed to generate appropriate synaptic inputs to motoneurons during different motor tasks.

Keywords: commissural cholinergic interneurons; motoneurons; muscarinic-dependent-short-term potentiation/modulation of synaptic transmission.

Figure 1

Ventral commissure stimulations induce both fast AMPA receptor-mediated EPSPs and slow muscarinic receptor-mediated EPSPs in lumbar motoneurons. (A) Schema of the spinal cord slice preparation (A1). Electrical stimulations of the ventral commissure (CS stim) were applied using tungsten bipolar electrodes. Motoneurons were recorded (Mn rec) in a whole-cell patch clamp configuration and filled with biocytin for post hoc identification [(A2), scale bar: 10 μm]. (B) Representative traces of the effect of a one shock, a 100-Hz, two shock and a 100-Hz, 20 shock CS stim recorded in current clamp conditions from a motoneuron in a strychnine/gabazine-containing saline (B1–B3) and with subsequent applications of DNQX (B4–B6) and atropine (B7–B9). a, b, and c represent the three different components of the response. Insets, traces at a higher time resolution of the initial part of the responses in control conditions (1) and in the presence of DNQX (2) (horizontal bar: 50 ms, vertical bar: 10 mV).

Figure 2

Atropine sensitivity of muscarinic receptor-mediated EPSPs and CS stimulation parameters. (A) Superimposed muscarinic (musc) EPSPs induced by a CS stim in the presence of different atropine concentrations (A1). Plot of the mean muscEPSP amplitude as a function of the atropine concentration (A2). Washout period: 30 min. (B) Representative traces of muscEPSP induced with different CS stimulation protocols (B1) in control conditions (black traces) and in the presence of atropine (200 μM, gray traces). (B2) Summary plot of the mean muscEPSP amplitude as a function of the number of shocks and frequency of the CS stim.

Figure 3

Commissural muscEPSPs are sustained by the closure of the potassium M-current. (A) Membrane input resistance changes were visualized by a rhythmic negative current application during the induction of muscEPSP (A1). (A2) Plot of the membrane input resistance as a function of time for the neuron presented in (A1). An increase in the input resistance was observed during the slow depolarization. (B) Representative traces of the CS stim in control conditions (black trace) and in the presence of the potassium M-current blocker XE991 [gray trace; (B1)]. The long-lasting part of the CS-stim-induced EPSP is suppressed, whereas the first part of the response is enhanced (B2). Representative traces of the effects of apamin (100 nM; gray trace) and apamin + XE991 on the whole CS HFS-induced depolarization (B3) and on the first part of the response (B4). (B5) Summary plot of the mean muscEPSP amplitude in the absence or presence of the different K⁺ channels blockers. The washout of XE991 was completed in 30–45 min.

Figure 4

Commissural muscEPSPs involve the activation of M2, M3, and M4 muscarinic receptors. (A) Representative traces of the effects of the M4-preferring antagonist, tropicamide (A1), and those of the M3-preferring antagonist, 4-DAMP (A2), on muscEPSP induced in control conditions (strychnine/gabazine/DNQX-containing aCSF) and after the washout of the drug (30–45 min). Summary plots of the mean muscEPSP amplitude (A3) and time constant (A4) as a function of the muscarinic antagonist concentration. (B) Traces illustrating the actions of 4-DAMP (B1) and pirenzepine (B2) on depolarizations induced by short-lasting applications of oxotremorine (oxo, 500 μM) in motoneurons synaptically isolated in a TTX/2 mM Mn²⁺-containing aCSF without Ca²⁺. (B3) Summary plot of the mean amplitude of the oxotremorine-induced depolarization in the absence or presence of XE991 and as a function of the different muscarinic antagonist concentrations tested.

Figure 5

High-frequency commissural stimulation induces muscarinic-dependent STP of commissural AMPA-EPSCs. (A) Application of a 100-Hz, 20 shock CS stim in current clamp conditions induced a STP in commissural (com) AMPA-EPSCs recorded at −60 mV (A1). Traces above the plot illustrate com-AMPA EPSCs recorded before (1), just after (2), and 500 s after (3) CS HFS. The middle panel traces show the CS HFS-induced response in current clamp conditions. (A2) Plot of the mean com-AMPA-EPSC area before and after CS HFS for all of the neurons tested. (B) The STP recorded in control conditions (black circles and traces) is blocked in the presence of atropine [gray circles and traces (B1)]. Traces above the plot illustrate com-AMPA-EPSCs recorded before (1), immediately following (2), and 500 s after (3) CS HFS. The middle panel traces show the inhibition of the long-lasting part of the CS HFS-induced response in current clamp conditions in the presence of atropine. (B2) Summary plot of the mean com-AMPA-EPSC area before and after the CS HFS in the absence (black circles) and the presence of atropine (200 μM, gray circles) for all of the neurons tested. (C) Representative traces of single shock CS stim-induced muscEPSCs (C1) recorded 360 s before (1), during (2), just after (3), and 540 ms after CS HFS (4) in a strychnine-gabazine-DNQX-containing aCSF. (C2) Plot of the mean muscEPSP area before and after CS HFS for all the neurons tested.

Figure 6

A STP of commissural AMPA-EPSCs could be induced with different stimulation paradigms of the ventral commissure. Time courses of the com-AMPA-EPSC area before and after a low-frequency stimulation of the ventral commissure [CS LFS (A)], a medium-frequency stimulation (MFS) of 20 shocks (B), of 40 shocks (C), and a high-frequency stimulation (D). A STP of com-AMPA-EPSC could be observed with the different stimulation protocols used.

Figure 7

Com-AMPA-EPSC STP is not altered by the potassium M-current antagonist, XE991, or the M3-preferring antagonist 4-DAMP. (A) Representative traces and time course of the com-AMPA-EPSC area before and after CS HFS in the control condition (black circles and traces) and in the presence of 10 μM XE991 [gray lozenges and traces (A1)]. Traces above the plot illustrate the com-AMPA-EPSCs recorded before (1), immediately following (2), and 600 s after (3) a CS HFS. The middle panel traces show that the CS HFS-induced responses in current clamp condition without and with XE991 in the superfusion medium. (A2) The average time course of the com-AMPA-EPSC area before and after the CS HFS in the absence (black circles) or the presence of XE991 (gray lozenges) for all of the neurons tested. (B) Representative traces and time course of the com-AMPA-EPSC area before and after CS HFS in the control condition (black circles and traces) and in the presence of 10 μM 4-DAMP [gray squares and traces (B1)]. Traces above the plot illustrate com-AMPA-EPSCs recorded before (1), immediately following (2), and 600 s after (3) CS HFS. (B2) Average time course of the com-AMPA-EPSC area before and after CS HFS in the absence (black circles) or the presence of 4-DAMP (gray squares) for all the neurons tested.

Figure 8

The muscarinic commissural transmission induced an STP of the contralateral lamina VII glutamatergic inputs but not of the ipsilateral lamina VII glutamatergic inputs. (A) Schema of the experimental procedure (A1). A tungsten electrode was placed on the ventral commissure (CS stim) and another in the ipsilateral lamina VII (ipsi lamina VII stim) to record ipsi lamina VII EPSCs from motoneurons (Mnrec) held at −60 mV, as exemplified by the trace in (A1). (A2) Plot of the mean ipsi lamina VII EPSC area as a function of time before and after CS HFS. No STP was induced in these experimental conditions. (A3) Plot of the mean com-AMPA EPSC area as a function of time before and after a CS HFS in five out of the eight neurons tested in the (A2) condition, showing the induction of com-AMPA EPSC STP in these neurons. (B). Schema of the experimental procedure (B1). Contralateral lamina VII EPSCs (contra lamina VII) were evoked in motoneurons as exemplified by the trace in (B1). (B2) The time course of the mean contra lamina VII EPSC area as a function of time before and after a CS HFS in the absence (black circles) or the presence of atropine (200 μM, gray circles).

Figure 9

Detection of cholinergic commissural interneurons in spinal cord slices. Photomicrographs of commissural interneurons (CINs) retrogradely labeled with Texas red (TxR), applied ventrally to the central canal and immunopositive for CholineAcetylTransferase (ChAT) in lamina VII (A) and close to the central canal (B). The areas boxed in (A,B) are shown at a higher magnification in (a,b) photomicrographs. Arrows point to the double-labeled CINs. (C) A normalized spinal cord transverse section illustrating the location of all the double-labeled CINs observed. The area boxed is shown at a higher magnification in the inset. [Calibration bars: (A–C), 200 μm; (a,b), 50 μm; inset, 100 μm].

Figure 10

Commissural muscarinic transmission transiently amplifies the motor output during fictive locomotion. (A) Schematic diagram of the experimental procedure. Fictive locomotion was recorded from the right lumbar 2, 5 and left lumbar 5 ventral roots (rL2, rL5, lL5) in the isolated spinal cord preparation. A tungsten bipolar electrode was used to stimulate the ventral commissure (CS stim). (B) Integrated recordings (I) of fictive locomotion induced by the bath application of NMA and 5HT (16 μM each) in the absence (B1) or presence of atropine [200 μM (B2)]. Circular analysis of the motor burst activity relationships (Φ) in the control condition (black circles and trace) and in an atropine-containing aCSF (white circles and dashed trace) (B3). (C) CS HFS (100 Hz, 20 shocks) induced transient activity in a quiescent spinal cord preparation in the control condition (C1) and in the presence of atropine (C2). (D). CS HFS application during NMA/5HT-induced fictive locomotion in the absence (D1) or presence of atropine (D2). (E) Summary plots of the normalized burst amplitude as a function of the burst number for the three-recorded ventral roots. Burst number −1 corresponds to the final complete burst recorded before the CS HFS, and burst number 1 corresponds to the first complete burst observed after the CS HFS. In the control conditions (black circles), CS HFS-induced a short-term potentiation of the amplitude of motor bursts recorded from the two L5 ventral roots that was occluded in the presence of atropine (white circles). (F) Summary plots of the fictive locomotion normalized burst amplitude as a function of the burst number for the three-recorded ventral roots before and after the application of a 100-Hz, 20 shock stim to the lL5 dorsal root (DR HFS).