Swimming against the tide: investigations of the C-bouton synapse

Abstract

C-boutons are important cholinergic modulatory loci for state-dependent alterations in motoneuron firing rate. m2 receptors are concentrated postsynaptic to C-boutons, and m2 receptor activation increases motoneuron excitability by reducing the action potential afterhyperpolarization. Here, using an intensive review of the current literature as well as data from our laboratory, we illustrate that C-bouton postsynaptic sites comprise a unique structural/functional domain containing appropriate cellular machinery (a "signaling ensemble") for cholinergic regulation of outward K(+) currents. Moreover, synaptic reorganization at these critical sites has been observed in a variety of pathologic states. Yet despite recent advances, there are still great challenges for understanding the role of C-bouton regulation and dysregulation in human health and disease. The development of new therapeutic interventions for devastating neurological conditions will rely on a complete understanding of the molecular mechanisms that underlie these complex synapses. Therefore, to close this review, we propose a comprehensive hypothetical mechanism for the cholinergic modification of α-MN excitability at C-bouton synapses, based on findings in several well-characterized neuronal systems.

Keywords: C-boutons; Kv2.1; SK; acetylcholine; afterhyperpolarization; subsurface cistern; α-motoneuron.

FIGURE 1

C-bouton synaptic sites contain a complex signaling ensemble. Presynaptic bassoon-IR and postsynaptic SK3-IR and m2-IR share a striking subsynaptic fenestrated appearance within the C-bouton. All images are small confocal stacks (3 × 1 μm Z-stacks) of en face C-boutons, indicated with VAChT-IR (blue), on rat lumbar α-motoneurons. (A) Presynaptic active zone protein bassoon (green) is aligned with postsynaptic ion channels SK3 (Ai, red) and m2 receptors (Aii, red). (B) Kv2.1-IR (green) intercalates with SK3-IR (Bi, red) and m2-IR (Bii), “filling in” the C-bouton postsynaptic membrane. Scale bars are 2.0 μm.

FIGURE 2

Synaptic distribution of specific ion channels and receptors on soma and proximal dendrites of motoneurons. The schematic illustrates three types of motoneuron presynaptic boutons including the glycinergic/GABAergic F-type, glutamatergic S-type and cholinergic C-type with its associated postsynaptic subsurface cistern. Note the specific localization of m2 muscarinic receptors (blue) with SK channels (red) and Kv2.1 channels (green) postsynaptic to the C-bouton. Small Kv2.1 clusters are also found postsynaptic to some S-type synapses (see Muennich and Fyffe, 2004). The P/Q- and N-type Ca²⁺ channels Cav2.1/2.2 (light gray) are illustrated throughout the membrane, although the precise subcellular localization of this channel is currently unknown. Both connexin 32 (pink) and the sigma-1 receptor (dark gray) are specifically associated with the C-bouton subsurface cistern.

FIGURE 3

The C-bouton synapse on mammalian α-motoneurons. (A) C-bouton synapses on intracellularly labeled and reconstructed adult rat lumbar α-MN are revealed by VAChT-IR (white). Large C-boutons densely innervate the soma and proximal dendrites of α-MNs but are absent from more distal locations. Also note that C-boutons are not located on motoneuron axons (indicated by “a”). (B) C-boutons, indicated by VAChT-IR (Bi,iv, white), are presynaptic to the muscarinic m2 receptor (Bii,iv, red) and large Kv2.1 clusters (Biii,iv, green). Note that m2 receptor immunoreactivity on the α-MN soma and proximal dendrites localize exclusively to C-bouton postsynaptic sites. (Bii) Inset shows subsynaptic fenestrated distribution of m2-IR. Images are confocal stacks of 12 × 1 μm Z-stacks with nissl stain (blue) to label adult rat neuronal somata. Scale bar is 20 μm. (C) Diagrammatic representation and electron micrograph of C-bouton ultrastructure in an adult rat. (Ci) Diagram illustrates densely packed, clear spherical or pleomorphic vesicles and abundant mitochondria. Closely apposed to the postsynaptic membrane is a 10–15 nm wide subsurface cistern (SSC) that is continuous with several lamellae of underlying rough endoplasmic reticulum (rER). Free ribosomal rosettes are typically visible in the subsynaptic region. (Cii) Electron micrograph of C-bouton synapse on an α-MN soma. Arrowheads indicate a SSC extending the entire appositional length of the bouton. Note key features present in electron micrograph illustrated in diagram (Ci).

FIGURE 4

The potassium ion channel SK3 is part of the C-bouton signaling ensemble in a subset of α-motoneurons. Images are confocal stacks of 26 × 1 μm Z-stacks with nissl stain (blue) to label rat lumbar neuronal somata. Scale bar is 20 μm. (A) VAChT-IR (white) C-boutons form synapses onto all rat lumbar α-MNs on the soma and proximal dendrites. (B) SK3-IR (red) located within surface membrane of a subset of α-MNs in large distinct clusters. In rodents, SK3 channels, having slower intrinsic activation and deactivation kinetics than SK2 channels (Xia et al., 1998), are preferentially expressed in small, presumably S-type, α-MNs with long duration and large amplitude mAHP currents (Deardorff et al., 2013). (C) Large and small Kv2.1-IR (green) clusters are located within the surface membrane of all α-MNs. (D,E) The large SK3-IR and Kv2.1-IR clusters colocalize within the surface membrane of α-MNs and are apposed to VAChT-IR C-boutons.

FIGURE 5

Subset of rat lumbar α-motoneurons with SK3-IR have significantly longer AHP 1/2 decay time and increased amplitude. Data shown is review of previous study reported by Deardorff et al. (2013). (A) Diagrammatic representation of experimental paradigms. In an adult in vivo rat preparation, tibial α-MNs, identified by antidromic activation of the tibial nerve, were penetrated with a sharp recording electrode. Neuronal electrical properties were recorded and neurons were filled with neurobiotin (green) for post hoc identification. Spinal cord tissue was harvested and processed for SK3-IR. (B–D) Neuronal electrical properties are of α-MNs depicted in micrographs below. Asterisk (*) denotes stimulus artifact. Micrographs are single optical confocal sections through the soma of intracellularly labeled α-MNs (green) processed for SK3-IR (red) and the general neuronal stain nissl (Blue). Scale bars are 20 μm. (B) SK3-IR (+) (Bii and Biii arrowheads) α-MNs have long duration and large amplitude AHP, low rheobase, and high input resistance. Micrograph insets show VAChT-IR (White) C-bouton in apposition to an SK3-IR (+) cluster. Inset scale bar is 5 μm. (C,D) SK3-IR (-) α-MNs have short duration and small amplitude AHPs. However, even among these SK3-IR (-) cells, rheobase and input resistance show high variance along the continuum of α-MN properties. Please note the nearby SK3-IR (+) cells (C,Dii,iii arrowheads).

FIGURE 6

Hypothesis for state dependent regulation of motoneuron activity through the C-Bouton signaling ensemble. (A) C-boutons increase motoneuron firing frequency along a widow of the α-MN activity spectrum. (Ai) With low or transient physiological drive, m2 activation is not likely to mediate an effect on AHP duration or firing rate. (Aii,iii) As excitatory drive increases, persistent m2 receptor activation inhibits local Ca_V channels through a G_i/G_o coupled pathway, preventing both the SK channel activation and Kv2.1 dephosphorylation. Thus, outward K⁺ current is reduced and neuronal firing rate is increased (relative to Bii and Biii) as illustrated with spike train below. (Aiv) m2-mediated effects on Ca_V channels are negated by prolonged or repeated membrane depolarization (Hille, 1994) as may occur during extremely strong or pathologic excitatory drive. Here, Ca²⁺ influx through N-type calcium channels activates SK channels to generate AHP and to dephosphorylate Kv2.1 to increase outward K⁺ current and reduce firing frequency, as illustrated with spike train below. (Bi–iii) As excitatory drive increases without C-bouton activity, the N-type Ca²⁺ influx activates SK channels to generate AHP. Thus, the outward K⁺ current maintains a lower firing frequency than in corresponding images in A. Spike trains illustrated below. (Biv) As in (Aiv), during prolonged or pathologic excitatory drive, N-type Ca²⁺ influx results in both SK channel activation and Kv2.1 dephosphorylation, thereby increasing outward K⁺ current and homeostatically decreasing firing rate, illustrated with spike train below. All spike trains depicted in this figure are added for illustrative purposes only and do not represent electrophysiological recordings or computer simulations.