Distribution of serotonin-immunoreactive cell bodies and processes in the abdominal ganglion of mature Aplysia.

Sensitization of the gill withdrawal reflex in Aplysia californica is an elementary form of learning, in part resulting from presynaptic facilitation of the LE mechanoreceptor neurons of the abdominal ganglion. It has previously been established that either application of serotonin or direct stimulation of a group of facilitatory neurons, the L29 cells of the abdominal ganglion, can simulate the effect of physiological stimulation in producing presynaptic facilitation. Because the evidence that serotonin serves as a facilitatory transmitter was indirect, we examined the distribution of serotonin-immunoreactive fibers and cell bodies in the abdominal ganglion in order to answer two questions: (1) do the sensory neurons receive serotonergic innervation and (2) are the L29 cells serotonergic? We observed two distinctive patterns of serotonergic innervation within the ganglion, sparse and dense. The sparse pattern is correlated with a serotonin-stimulated increase in cAMP in identified target cells, while the dense innervation is not. We found a sparse distribution of serotonin-immunoreactive fibers with varicosities close to both cell bodies and processes of identified LE sensory cells. It therefore is likely that the sensory neurons do receive serotonergic innervation. We also mapped the population of serotonergic neuronal cell bodies in the ganglion, and found five clusters of neurons. Cells in one of these clusters, the identified RB neurons, had previously been shown to synthesize serotonin from tryptophan and to contain the neurotransmitter in high concentration. Identified L29 facilitator cells marked by injection with Lucifer Yellow do not contain serotonin immunoreactivity and therefore evidently are not a source of serotonergic input onto sensory cells.

Distribution of serotonin-immunoreactivity in juvenile Aplysia.

Serotonin-immunocytochemistry has been applied to whole mounts of the central nervous system and of several peripheral tissues from stage 12 juvenile Aplysia californica. The small size of animals at this stage permits visualization of the three-dimensional distribution of structures containing serotonin-immunoreactivity in unsectioned tissues. Many neuronal cell bodies are stained in addition to the giant cerebral neuron of the cerebral ganglion and cells in the RB cluster of the abdominal ganglia which previously had been characterized biochemically and pharmacologically as being serotoninergic. Neuronal cell bodies, both in central ganglia and in the wall of the gut, are encircled by plexuses of serotoninergic varicosities. The neuropil of ganglia and the eye also contain fine, immunoreactive axons bearing varicosities. Intraganglionic connectives and nerves contain many stout fluorescent axons. Serotoninergic varicosities are also observed in the connective tissue sheath surrounding central ganglia and nerves, as well as in heart and body muscle, blood vessels and gut.

Retrograde axonal transport of a wheat germ agglutinin-horseradish peroxidase conjugate in Aplysia californica.

To begin examining retrograde transport in a single invertebrate neuron we have used wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP), 18 h after the conjugate was injected extracellularly into the neuropil of the buccal ganglion of Aplysia, HRP reaction product could be detected cytochemically in putative lysosomes, cisternae and vesicles near Golgi in the giant metacerebral neuron (GCN) of the cerebral ganglion. Appearance of reaction product in the cell body was blocked by ligating GCN's axon and was inhibited by colchicine.

Penetration of horseradish peroxidase into the optic nerve after vitreal or vascular injections in the developing chick.

In order to determine whether a possible barrier exists to diffusion of tracer into the optic nerve during development and to provide a basis for later studies of retrograde axonal transport in embryonic nerves, we studied the diffusion of horseradish peroxidase (HRP) into the nerve after vitreal injections in chicks ranging in age from embryonic day 6 to 3 days after hatching. We found that HRP may reach the periaxonal spaces of the retrobulbar optic nerve after vitreal injection, vitreal injection into the opposite eye, or vascular injection. These and other observations suggest that vitreally injected HRP may reach the periaxonal spaces of the optic nerve by at least two routes: (1) by the obvious diffusion of marker from the vitreal surface into the optic nerve head and (2) by vascular leakage from fenestrated capillaries of the choriocapillaris into the pericapillary spaces of these and other capillaries that feed into the optic nerve parenchyma. There is a breakdown in the blood-brain barrier to HRP in the optic nerve head of the chick at embryonic day 13. The development of the breakdown depends at least in part on the maturation of vasculature in the nerve and the establishment of anastomotic branches between these vessels and those of the choriocapillaris. Our results further suggest that the limited diffusion of HRP into the retrobulbar nerve of fetal and newly hatched chicks is a function of uptake of tracer by glial cells within the nerve. Investigators of axonal transport who use this visual pathway as a model should be reminded of the potential artifact involved in this access of vascularly circulating label into the region of the lamina cribrosa.

Uptake and anterograde axonal transport of wheat germ agglutinin from retina to optic tectum in the chick.

The uptake and anterograde axonal transport of 125I-wheat germ agglutinin (WGA) has been investigated in the visual system of the chick. In order to obtain a marker with specific and homogeneous binding properties, the iodinated lectin was affinity purified by passage over an N-acetylglucosamine (NAcGlu)-Sepharose column after iodination. 22 h after vitreal injection of the purified 125I-WGA, radioactive label was found accumulated in the retinoreceptive layers of the contralateral optic tectum. Gel electrophoresis of tectal homogenates revealed that greater than 80% of the retrieved label ran in a band which comigrated with native WGA. In chicks injected with the fraction of the iodinated preparation that failed to bind to the affinity column, there was no evidence of tectal labeling. These findings support the hypothesis that WGA is selectively taken up by chick retinal ganglion cells and transported intact in an anterograde direction to their axon terminals in the contralateral optic tectum. This raises the possibility that constituents of perikaryal membrane, i.e., lectin receptors, are transported in an anterograde direction by chick retinal ganglion cells.