Melvin J. Cohen

Branching of Central Neurons: Intracellular Cobalt Injection for Light and Electron Microscopy

Cobalt chloride can be injected into an identified nerve cell body in an insect ganglion and reacted with ammonium sulfide to stain the soma and its branches with a black precipitate. The stained cell body and its branches throughout the neuropil are visible in both the light and electron microscope. In whole mount preparations, the resolution of neurites within the neuropil is of a quality that permits the comparison of branching patterns between cells and during various functional states.

Synaptic regeneration and glial reactions in the transected spinal cord of the lamprey

SummaryWe have examined axonal growth and synaptic regeneration in identified giant neurons of the transected lamprey spinal cord using intracellular injection of horseradish peroxidase. Wholemounts together with serial section light and electron microscopy, show that axons from identified Müller and Mauthner reticulospinal neurons grow across the lesion and regenerate new synaptic contacts. Relatively normal swimming returns in these animals by 3–4 weeks after spinal transection. This occurs despite the formation of regenerated synapses in regions of the cord that are not usually occupied by these neurons.The regenerating axons branch profusely in contrast to their unbranched state in the normal animal. In addition to showing the two synaptic configurations found normally, synapses may be formed by slender sprouts from the growing giant axon. These ‘sprout’ type synaptic contacts appear unique to the regenerating neuron. Only regenerated chemical synapses were seen; the morphologically mixed chemical and electrical (gap junction) synaptic complex common in the normal animal was not observed at regenerated synapses.The site of spinal transection in the functionally recovered animal shows an increase in the number of ependymal and glial cells. Ependymal-like cells appear in regions away from the central canal. The expanded ependymal and glial processes covering the peripheral surface of the injured cord become convoluted, in contrast to their normal smooth configuration. There is no collagen within the cord at the site of transection but a considerable deposition is seen external to the cord surface.Axonal growth across a spinal lesion and subsequent synaptic regeneration can be examined in single identifiable giant interneurons in the spinal cord of the larval lamprey. This preparation may be used as an assay to investigate factors that could contribute to functional recovery following central nervous system injury in the higher vertebrates.

Electrical Responses of Insect Central Neurons: Augmentation by Nerve Section or Colchicine

Intracellular recording from the somata of central motor neurons in the cockroach Periplaneta americana normally shows little or no electrical response evoked by soma depolarization or by antidromic stimulation. Within 4 days after either cutting the axon or administration of colchicine, large action potentials can regularly be recorded from cell bodies of metathoracic motor neurons. Each experimental procedure evokes formation of a dense, perinuclear ribonucleic acid ring in the soma of neurons showing augmented electrical responses.

Two types of presynaptic configurations in insect central synapses: an ultrastructural analysis.

Two structurally distinct types of synapses have been identified in the cockroach metathoracic ganglion. The two synaptic types are distinguished on the basis of (a) the shape and position of the presynaptic density seen by serial thin sectioning and (b) the arrangement and location of vesicle attachment sites (VAS) on the presynaptic membrane obtained from replicas of aldehyde-fixed, freeze-fractured neuropile. Bar-type synapses in thin sections possess a long presynaptic density located in a trough or groove opposite the extracellular space between two contiguous postsynaptic processes. In freeze-fracture, this trough is flanked by two rows of vesicle attachment sites. The second synaptic conformation consists of rows of discrete dense projections located on the convexities of the presynaptic membrane, i.e., directly opposite a single postsynaptic process. This conformation has been correlated with groups of VAS linearly arranged also on the convexities of the presynaptic membrane. These structurally different synapses may represent functionally different contacts within the insect ganglion.

The Form of Nerve Cells: Determination by Cobalt Impregnation

Form and function are perhaps more closely related in neurons than in any other cell type. A major approach to understanding the cellular events underlying behavior concentrates on relatively simple invertebrate systems that give rise to specific behavioral acts. The general procedure followed is (1) to identify the individual neurons that generate the behavior, and (2) to analyze the particular pattern of connections between neurons to determine the properties of the circuit that might directly govern the form of the behavioral act. Initial studies dealt primarily with identification of nerve cell bodies because of their relative accessibility. This led to the production of neuron soma maps for several invertebrates (Hughes and Tauc, 1962; Otsuka et al., 1967; Nicholls and Baylor, 1968). One such cell body map for the cockroach is shown in Fig. 1 (Cohen and Jacklet, 1967). The distribution of about 50 bilateral pairs of motoneurons is defined in relation to the nerve trunks through which their axons leave the ganglion.

Glycogen in the central neurons of insects: Massive aggregations induced by anoxia or axotomy

Abstract Large nerve cell bodies in the metathoracic ganglion of the cockroach, Periplaneta americana were examined for glycogen content. Periodic Acid Schiff (PAS) staining of frozen sections, together with electron microscopic evidence, confirmed the low glycogen content in normal neuron somata. Subjecting animals to a variety of stressful factors including axotomy and CO 2 or N 2 anaesthesia resulted in a rapid and substantial increase in neuronal glycogen. The glycogen appeared as large aggregates 1–3 μm in diameter closely associated with numerous mitochondria. These structures were principally restricted to the trophospongial regions of the nerve cell cytoplasm. Glycogen was rarely encountered in the trophospongial glia either in normal or experimental animals. The glycogen response, which persisted in large nerve cell bodies for at least 6.25 days, is discussed in relation to fluctuations in circulating trehalose resulting from the rapid mobilization of fat body glycogen.

TROPHIC INTERACTIONS IN EXCITABLE SYSTEMS OF INVERTEBRATES

The nature of trophic relationships has been extensively discussed in the present volume, and it is sufficient to state here that we are dealing with a problem in relatively longterm communication. The communication may be between excitable cells, but it may also occur between parts of a cell, such as the axon and soma. The time scale approximates the life span of the individual or, at the least, the life of the cells involved, in contrast to the transient millisecond to millisecond span associated with the events of neural information processing. The wealth of material on trophic function collated in this volume has been systematically acquired on a relatively small number of vertebrate preparations. These studies draw heavily upon the classical background of vertebrate anatomy, physiology, and biochemistry. In nloving from this vertebrate framework, with its mass of supporting data, to the area of invertebrates, one must immediately ask what advantages are to be gained. The phylogenetic spread imposes great variability on invertebrate material, and supporting data is often scarce or nonexistent. Against &ese rather significant hazards, the critical advantages offered by invertebrate preparations for the study of trophic problems can be listed as follows: (1) the ability to work with single identified cells or even parts of a cell; (2) the extreme anatomical and functional stabilty of the material: and (3) the ability to interpose precise and exag gerated changes against this background of stable structure and function. Information bearing on trophic problems is scattered and relatively scarce in the invertebrates, and it is not my purpose here to attempt a systematic review of this material. Rather, I propose to discuss and document the particular advantages mentioned above in the light of recent developments.

Changes in firing behaviour of an insect motor neurone during development and following experimental surgery

Abstract The firing behaviour of an identified neurone in the cricket was studied using extracellular recording from the axon. In the last nympal instar (preadult developmental stage), the contralateral dorsal longitudinal motor neurone (CDLM) showed spontaneous activity and was excited by air puffs to the head and cerci and by single shocks to the anterior nerve cord. In the normal adult the CDLM did not exhibit these properties. However, responses which were characteristic of the last instar appeared in the adults which had been subjected to any one of the following surgical procedures: (1) central nervous system injuries which separated the CDLM arborization and axon from the soma; (2) operations which injured the central nervous system without cutting the CDLM; and (3) operations which damaged the cuticle only. Since cuticle damage alone was as effective as the more extensive operations, it is suggested that a sufficient cause for the appearance of nymphal firing behaviour in the adult CDLM is cuticle damage. The factor associated with cuticle damage which mediates the changes in activity of the CDLM neurone is not known, but its action does not require the mediation of the CDLM soma.