0006-Ch02.pdf

Spinal Motoneurons:

Synaptic Inputs and

Receptor Organization

Robert E. W. Fyffe

CONTENTS

2.1 Introduction

2.2 Synaptic Terminals on Motoneurons

2.2.1 F-Type Boutons

2.2.1.1 Colocalization of Glycine and GABA at Ventral

Horn Synapses

2.2.2 C-Terminals

2.2.3 S-Type Boutons

2.2.3.1 Synapses Formed by Aminergic and Peptidergic

Terminals on the Soma of Motoneurons

2.2.4 Summary

2.3 Diversity of Synaptic Terminal Structure: Functional Implications

2.3.1 Relationships between Ultrastructural Features

at Central Synapses

2.4 Passive and Active Properties of Dendrites

2.4.1 Voltage-Dependent Channels in Dendrites

2.5 Distribution of Synaptic Inputs

2.5.1 Ia Afferent Fibers

2.5.1.1 Distribution of Glutamate Receptors

2.5.2 Corticospinal Axons

2.5.3 Vestibulospinal Axons

2.5.4 Inhibitory Inputs from Renshaw Cells and Ia

Inhibitory Interneurons

2.5.4.1 Distribution of Receptors for GABA and Glycine

2.5.5 Serotoninergic Inputs

2.5.5.1. Distribution of 5-HT Receptors

2.5.6 Are Synaptic Inputs Compartmentalized?

References

2.1 INTRODUCTION

Spinal motoneurons have long been the focus of studies of synaptic action and mem-

brane properties, and the general ﬁring properties and morphological features of both

alpha and gamma motoneurons are well-known. 1 In a variety of mammalian and non-

mammalian vertebrate species both types of motoneurons are intermingled in topo-

graphically organized elongated columns of cell bodies in lamina IX. 2–6 Whereas moto-

neuron cell bodies are positioned in circumscribed locations or “pools”, their dendritic

trees exhibit complex branching architecture. Archetypically, alpha motoneurons inner-

vating hindlimb muscles in the cat have radially organized dendrites, with profuse

extensions throughout the ventral horn and adjacent white matter. These dendritic trees

are relatively symmetrical, although regions just ventral to or dorsal to the soma have

fewer dendritic projections than expected. Dendritic trees of gamma motoneurons inner-

vating the same muscle groups are generally less symmetrical, have fewer branch points

and less total membrane area, but have dendrites that can be just as long as those of

alpha motoneurons. Notably, alpha motoneuron dendritic morphology deviates from

the radially symmetric pattern in a number of motoneuron pools, such as those inner-

vating neck muscles or muscles of respiration, where dorso-medial and dorso-lateral

bundles of dendrites are prominent. Detailed quantitative light microscopic descriptions

of various alpha and gamma motoneurons are available. 7–22

Throughout the nervous system, the critical role of the dendrites in neuronal

function is emphasized by the fact that they provide more than 95% of the available

membrane surface area for reception of synaptic contacts; for alpha motoneurons,

this receptive area must accommodate approximately 50,000 synaptic inputs from

a variety of segmental and supraspinal sources. 23 Despite broad recognition that

neuronal dendrites have active properties (see below), there is consensus that the normal

site of action potential initiation is at the axon initial segment or ﬁrst node of Ranvier

on the axon, even though under certain input conditions action potentials can be initiated

in the dendrites 24–26 (see also, Reference 27). Ultimately, therefore, cell ﬁring is depen-

dent on the amount of current that reaches the cell soma/initial segment from active

synapses that are widely distributed over the dendritic surface. 1,28–31

Thus, although both presynaptic and postsynaptic factors play critical roles in

synaptic efﬁcacy per se, the amount of current delivered to the soma, and hence the

overall integrative properties and general excitability of a neuron, is strongly inﬂu-

enced by the interaction of postsynaptic factors such as intrinsic membrane properties

and the distribution and density of receptors and ion channels over the somato-

dendritic membrane as well as by the spatial distribution of the synapses on the

complex branching dendritic tree. From an historical perspective the study of moto-

neuron dendrites has been particularly inﬂuential in promoting these concepts and

this chapter will review some issues of continuing relevance to neuronal integration,

with particular focus on spinal motoneurons that innervate skeletal muscles.

2.2 SYNAPTIC TERMINALS ON MOTONEURONS

The synaptic terminals that are present on the motoneuron soma (and dendrites) can

be classiﬁed into a relatively small number of categories based on ultrastructural

criteria such as the size of the terminal, the shape of the synaptic vesicles contained

in the bouton, and the disposition of the postsynaptic densities at the synaptic

junction. 32–34 In general, four main types (S-, F-, C-, and M-type boutons; see

Figure 2 in Reference 33) of boutons are described on alpha motoneurons, although

modiﬁcations to the classiﬁcation scheme have been introduced by subsequent

authors 23,35 (see also, Figure 3.4 in Reference 1), in part because contemporary

labeling techniques used to visualize identiﬁed motoneurons or boutons may obscure

some of the deﬁning features in the pre- or postsynaptic elements of the synapse.

Because postsynaptic structural elements can be obscured in this way, synaptic

vesicle shape (and distribution of vesicles within the terminal) and bouton size were

used as the main classiﬁcation factors in intracellular staining studies, resulting in

use of a tripartite system encompassing F-type (which contain pleomorphic synaptic

vesicles) and two categories of S-type (small and large boutons with spherical

synaptic vesicles) boutons. An additional bouton type, the P bouton, makes presyn-

aptic connections with some of the other bouton types in contact with the motoneuron

surface and may form triadic arrangements. 33,36

Detailed synaptological studies have attempted to determine the frequency of

each bouton type on different areas of the surface membrane, and where possible,

their origin. The task of systematically surveying the complete motoneuron surface

is daunting and unrealistic, if not impossible, because the surface area of an alpha

motoneuron often exceeds 5

10 5

m 2 , most of which is dendritic membrane, and

m. Electron microscopic studies can only sample

a very small fraction of that area, and typically, a detailed study can only be expected

to sample from at most a few neurons. Quite justiﬁably, quantitative electron micro-

scopic studies of bouton frequency and packing density have focused on the somatic

membrane, and to a lesser extent on selected dendritic regions at various distances

from the soma. Estimates of overall bouton frequency and total number of synapses

are then based on extrapolation of data from these sampled regions.

Identiﬁcation of motoneuron somata for the purposes of ultrastructural analysis

may be based on intracellular labeling, on the transport of markers from the periph-

ery, or on soma size and location in the ventral horn. Although primary dendrites

are easily recognized as they emerge from the soma, ultrastructural analysis of

identiﬁed thin distal dendrites requires the use of intracellular staining strategies and

subsequent light–electron microscopic correlation of selected labeled proﬁles. Cou-

pled with 3-dimensional reconstruction of dendritic paths, this approach provides

an accurate measurement of distance from the soma. Even if labeled dendritic proﬁles

are not rigorously connected to a full reconstruction of an individual neuron their

approximate position can be estimated if their diameter is known, because of the

fairly consistent relationship between dendrite diameter and distance that has been

revealed by quantitative light microscopic studies of neurons reconstructed in three

dimensions. 37 Surprisingly, in view of the inherent technical difﬁculties and the

uncertainties about conclusions drawn from “small” samples (which may actually

represent thousands of electron micrographs and measurements), there is a growing

consensus from quantitative ultrastructural studies that spinal alpha motoneurons

receive approximately 50,000 synaptic boutons over their total somatic and dendritic

surface membrane. 23 Other recent studies have placed the number of synapses in the

total dendritic lengths exceed 10 5

m 2 of somatic mem-

brane, thus suggesting that there may be around 500 to 1000 synapses on each alpha

motoneuron cell body. This represents only 1 to 2% of the total number of synaptic

inputs on each motoneuron.

Despite the well-recognized problems associated with sampling and classifying

bouton types, elegant quantitative ultrastructural analyses have also reached a rela-

tively strong consensus regarding the frequency of boutons on the soma surface and

on the proportion of surface membrane occupied by the various synaptic types. For

cat and primate alpha motoneurons, the percentage of somatic membrane covered

by bouton proﬁles is usually around 50% (range 40 to 60%) 23,33,34,38–41 but may be

as high as 70% in neck motoneurons. 35 In contrast, gamma motoneurons exhibit

fewer somatic synapses and a lower (20 to 40%) level of membrane coverage. 40,42,43,45

In alpha motoneurons, the percentage of proximal dendritic membrane in contact

with axon terminals is generally about the same, or slightly higher, than for the

soma. However, dendritic membrane coverage declines markedly at greater distances

from the soma, to levels around 20 to 30% or less. 23,35 The frequency of occurrence

of different bouton types also varies from cell to cell and in different regions of the

dendritic tree, as discussed in the following sections.

2.2.1 F-T YPE B OUTONS

F-type boutons are the most numerous type of synapse on the somatic surface of

alpha motoneurons, comprising more than half (usually 50 to 60%) of the contacts

at the soma, and accounting for a similar proportion of the synaptic membrane

coverage. F-type bouton frequency remains relatively high, comparable to somatic

levels, on the proximal dendrites but declines on more distal dendrites. F-type

boutons contain pleomorphic (elongate or irregular shape) synaptic vesicles, usually

form symmetric synaptic junctions, and are generally assumed to represent inhibitory

terminals. In keeping with this assumption, boutons of this class arising from iden-

tiﬁed inhibitory interneurons, 44 and F-type boutons in contact with the somata or den-

drites of presumed motoneurons, 37,40,46 are enriched with glycine-like immunoreactivity.

2.2.1.1 Colocalization of Glycine and GABA at Ventral

Horn Synapses

Glycine and GABA are the two major inhibitory amino acid transmitters in the

spinal cord and are involved in a variety of inhibitory actions on motoneurons. A

number of speciﬁc antibodies that were developed to reveal the presence of GABA,

glycine, and their respective receptors and vesicular transporters, have been used

extensively in investigations of inhibitory inputs to motoneurons in cat and rat

spinal cord. 37,39,46–53 These studies substantiate the link between F-type bouton

range of 50,000 to 140,000 on alpha motoneurons. 37 In the following discussions it

is tacitly assumed that the value is closer to the lower end of that range, i.e.,

approximately 50,000 synapses per motoneuron. Although it is difﬁcult to deﬁne

accurately the area of the soma surface, 11 various quantitative electron microscopic

studies provide estimates of around 10 to 12 synapses per 100

morphology and an inhibitory role for this type of synapse, and importantly, have

helped to consolidate the idea that classical amino acid neurotransmitters may

coexist and act in concert at fast central synapses. It is now clear that motoneurons

receive inputs from glycine, GABA, and mixed glycine and GABA synapses in

various proportions.

One commonly used marker of presumed inhibitory synapses is gephyrin, which

is a peripheral membrane/cytosolic protein involved in the clustering and/or stabi-

lization of postsynaptic receptors at glycinergic and GABAergic synaptic sites. 54,55

On motoneuron cell bodies the vast majority (approx. 90%) of synapses formed by

F-boutons exhibit postsynaptic gephyrin immunoreactivity. 52 Interestingly, in Ren-

shaw cell interneurons a very high proportion (around 90%, compared to 50 to 60%

on alpha motoneurons) of all somatic synapses are formed by F-boutons, and vir-

tually all of these synapses (96%) are immunopositive for gephyrin. 52

Although initially discovered in association with glycine receptors, and known

to be tightly associated with membrane-spanning subunits of the glycine receptor

in spinal cord synapses, especially in the ventral horn, 48,56–58 gephyrin is also involved

in the clustering and/or stabilization of GABA A receptors in many brain regions,

including the spinal cord. 57,59,60 A major challenge in recent years has been to

untangle the intricate relationships between the molecular components that underlie

inhibitory transmission mediated by glycine and GABA in the ventral horn of the

spinal cord; in this effort, the detailed studies from the laboratories of F.J. Alvarez,

A.J. Todd, and A. Triller have been particularly illuminating. 57,58,61–63 Related studies

of retinal synapses have provided similar ﬁndings. 64 Cumulatively, the studies of

motoneurons and Renshaw cells indicate that

1. Immunoreactivity for glycine receptors and certain GABA A receptors is

colocalized with gephyrin at synaptic specializations in the spinal cord.

2. Virtually all (90 to 98%) of the gephyrin-immunoreactive postsynaptic

sites are associated with glycine-enriched presynaptic terminals; a signif-

icant minority (around 30 to 35%) of the terminals also contain GABA

(this proportion is much higher on Renshaw cells); very few (around 2%)

of the presynaptic boutons contain only GABA.

3. Glycine receptors and GABA A receptors are colocalized in at least two thirds

of the gephyrin-labeled synapses; of the remainder, the majority have glycine

receptors exclusively, and only 2 to 3% have GABA receptors exclusively.

4. There are many synapses on motoneurons and Renshaw cells at which

both glycine and GABA are enriched presynaptically, and at which both

glycine and GABA A receptors (and gephyrin) are present postsynaptically.

Although interpretation of these results is complicated (for example, they suggest

the existence of some synapses at which GABA A receptors are present postsynap-

tically, but GABA is not in the apposed presynaptic bouton), the signiﬁcance of

mixed glycine–GABA synapses is emphasized by functional conﬁrmation that core-

lease of GABA and glycine occurs at synapses of spinal neurons. 65

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