Kenneth J. Muller

The Shapes of Sensory and Motor Neurones and the Distribution of their Synapses in Ganglia of the Leech: A Study Using Intracellular Injection of Horseradish Peroxidase

Three types of sensory neurones and two kinds of motor neurones in the segmental ganglion of the leech were examined with the light and electron microscope after intracellular injection of horseradish peroxidase (HRP) for a histological marker. The aim was to develop a method for identifying the synapses of specific cells in the ganglion’s complex neuropil and to form a picture of their distribution and structure. Reaction of HRP with different benzidine derivatives produces opaque and electron dense deposits. For light microscopy a blue stain is formed that makes processes visible in whole mounts millimeters away from the injection site at the soma. The reaction product for electron microscopy is distributed throughout the cytoplasm, yet ultrastructural details are preserved. The sensory neurones that respond specifically to touch or pressure or noxious mechanical stimuli to the skin share in their branching pattern a number of common features. A single process arising from each cell body forms large primary branches that pass through the neuropil and leave the ganglion by the ipsilateral connectives and roots. Within the neuropil these branches give rise to numerous smaller secondary processes. In contrast, the annulus erector and large longitudinal motoneurones send their main process across the ganglion to bifurcate and enter the contralateral roots. Secondary processes of the motoneurones are highly branched and more numerous than those of the sensory cells. Each type of sensory and motor cell is distinguished by the shape, length and distribution of its secondary processes. Secondary processes of sensory neurones exhibit numerous swellings and irregularly shaped fingers. Electron micrographs show that the sensory neurones make synapses at these specializations, each of which contacts several postsynaptic processes. The sensory neurones receive inputs at the same fingers and swellings, an arrangement suggesting that regions within a cell’s arborization may function semi-autonomously. The main process and large branches of the two motor neurones are studded with spines a few micrometres long and a fraction of a micrometre in diameter. Vesicle-containing varicosities from other cells make synaptic contact primarily with the spines, which themselves have few vesicles. These two motor neurones are largely, if not entirely, postsynaptic to other neurones within the leech nervous system.

Synapses between neurones in the central nervous system of the leech.

CONTENTS

Microglial movement to sites of nerve lesion in the leech CNS

The small glial cells in the central nervous system of the leech, Hirudo medicinalis, have been studied using two histological stains. Weak silver carbonate, a classic stain for vertebrate microglia, can selectively stain these small glial cells and shows that they are morphologically similar to vertebrate microglia. Feulgens DNA-specific stain is useful for counting the compact and distinctive microglial nuclei. In uninjured connectives, which link segmental ganglia, there are 134 +/- 28 microglia per 210 micron of connective length. Within 24 h after the nerve cord is crushed leech microglia aggregate at the site of injury. This increase in cells, seen both in vivo and in culture, is approximately 5-fold. Although cells do not continue to accumulate at the injury site after the first day, their numbers continue to vary with time in the regions immediately adjacent to the crush for at least one week. A second crush made 24 h after the first shows that leech microglia are capable of responding to repeated injury.

Morphological evidence that regenerating axons can fuse with severed axon segments

Regenerating axons of sensory neurons in the leech nerve cord usually reconnect with their normal targets by growing the entire distance from the site of lesion to the target. However, in less than 1% to nearly 10% of cases a rapid restoration of the normal arborization occurs when the regenerating axon connects with the severed distal segment of the same cell or another cell of the same modality. The passage of horseradish peroxidase (mol. wt approximately 40,000 daltons) from the regenerating axon selectively into the axon or cell with which it has connected indicates that the two have joined or fused, rather than become linked by an electrical synapse, as sometimes occurs for other neurons in the leech. These results support the conclusions, based largely on physiological data from regenerating motor axons in crayfish, that unusually rapid and complete regeneration can occur when a growing axon fuses with its severed distal segment.

Mapping of neuronal contacts with intracellular injection of horseradish peroxidase and Lucifer yellow in combination

A technique for the simultaneous visualization in the light microscope of processes of neurons filled with horseradish peroxidase and Lucifer yellow in combination has been developed. The technique is applied to determine the location, number and distribution of presumptive synaptic sites between neurons in the leech central nervous system.

Chapter 2 Nerve Fiber Growth and the Cellular Response to Axotomy

Publisher Summary This chapter explores some of the complex events within the nerve cell that are triggered by axotomy—the severing of the axon. It emphasizes the interconnectedness of results obtained in a wide variety of systems, from functioning mammalian central and peripheral nervous systems to embryonic cells regenerating in culture and invertebrate nervous systems. It also examines what changes appear in the axotomized neuron. Once the transition from injury to regeneration occurs, regenerating neurons acquire many of the specializations of developing neurons. The movement of the growth cone and elongation of the axon are dependent upon the cells synthetic machinery and require a specialized cytoskeletal, contractile, and transport apparatus whose mechanisms are not well understood. Because some basic mechanisms of neuronal growth might be expected to be shared with nonneuronal motile cells, the chapter examines growth, metabolism, and cytoskeleton on a more general scale.

Long-term survival of glial segments during nerve regeneration in the leech.

Nerve injury that severs axons also disrupts ensheathing glial cells. Specifically, crushing or cutting the leech nerve cord separates the glial cells nucleated portion from an anucleate recording, by intracellular injection of Lucifer Yellow dye and horseradish peroxidase (HRP) as tracers, and by electron microscopy. The nucleated portion of the glial cell did not divide, degenerate, or grow appreciably. The severed glial stump remained isolated from the nucleated portion but maintained its resting potential and normal morphology for months. Stumps typically began to deteriorate after 3 months. Small macrophage-like cells, or microglia increased in number after injury and ensheathed axons, thus partially replacing the atrophying glial stump. Some axons in the nerve cord degenerated; the remainder appeared morphologically and physiologically normal. Thus, both nucleated and anucleate glial segments persisted throughout the one to two months required for axons to regenerate functional connections. Glial cells in the leech are therefore available to guide physically the growing axons or to contribute in other ways to nerve regeneration.

Synapse regeneration and signals for directed axonal growth in the central nervous system of the leech

In each segmental ganglion of the leech there is one S interneuron which extends an axon anteriorly and posteriorly halfway along the connectives to make electrical synapses exclusively with the tips of the next S-cell axons. Fluorescent dyes of low molecular weight such as Lucifer Yellow [Stewart, W. W. (1978). Cell 14, 741–759] pass selectively from one S-cell axon to the next. An S-cell axon that has been severed regenerates along its distal stump to synapse with its usual target, the adjacent S-cell axon [Muller, K. J., and Carbonetto, S. T. (1979). J. Comp. Neurol. 185, 485–516]. In experiments reported here, Lucifer Yellow injected into a regenerated S cell passes exclusively into the target S cell, indicating that the injured cell regenerates a synapse specifically with its usual target. When the target of a severed S-cell axon is selectively eliminated by the intracellular injection of protease, the severed axon regenerates along its distal stump in a normal fashion, stopping at the usual site of synapse without making detectable alternative connections. Similarly, intact S cells are apparently unaffected by the protease killing of their target; they neither grow nor retract and form no aberrant synapses. In contrast, when its target is killed, an intact axon can be triggered to sprout at its tip by injuring another branch of the same cell. The sprouted axons grow along the pathway formerly occupied by the killed cell and make no detectable synapses. It appears that cellular injury turns on general growth processes within the neuron that are expressed only by axonal branches that have lost contact with their target.

Distinctions between gap junctions and sites of intermediate filament attachment in the leech C.N.S.

SummaryFreeze-fracture studies on the nerve cord of the leechHirudo medicinalis reveal that the plasma membranes of various cells, including glial and muscle cells, contain at least two distinct types of aggregated intramembrane particles, identified as hemidesmosomes and gap junctions. Hemidesmosomes consist of angular particles irregularly arranged in circular or elongate patches in external leaflets (E-faces), and are associated with a bundle of intermediate filaments extending into the cytoplasm. Hemidesmosomes of specific axons abut on extracellular space at openings in the surrounding glial sheath. Gap junctions are patches of rounder particles in cytoplasmic leaflets (P-faces) and are more uniformly spaced; they have a corresponding array of pits in the complementary E-face. Gap junctions connect processes of adjacent smooth muscle cells, and apparently interconnect glial processes. Thus, different types of cells in the leech C.N.S. have similar intramembrane specializations. Moreover, the hemidesmosomes and gap junctions might, on superficial examination, be confused.