Immunoreactivity in Limulus: III. Morphological and biochemical studies of FMRFamide‐like immunoreactivity and colocalized substance P‐like immunoreactivity in the brain and lateral eye

Abstract

FMRFamide-like immunoreactivity (FLI) and the colocalization of FMRFamide and substance P-like (SPLI) immunoreactivities were examined in the brain and lateral eye of the horseshoe crab with FITC- and TRITC-labeled secondary antibody techniques. In the brain, fibers with FLI were localized in the neuropils of the lamina, medulla, central body, corpus pedunculatum, optic tract, circumesophageal connective, and central neuropil. An extensive network of reactive fibers innervatives the brain's vascular sheath. Somata with FLI were found in the dorsal medial group, dorsal lateral posterior groups #1 and #2, and ventral posterior lateral groups #1 and #2. Several distinct subgroups of reactive somata were noted in both the medullar and ventral medial groups. The distribution of fibers in the brain with colocalized FLI and SPLI includes those which innervate the vascular sheath and widespread populations of small-diameter beaded fibers in the central neuropil and circumesophageal connective. Somata with colocalized FLI and SPLI constitute minority populations in the medullar and dorsal medial groups but form the majority population of a subgroup in the ventral medial group. Overall localization of SPLI was reevaluated and is reported here according to the nomenclature of the new Chamberlain and Wyse brain atlas. In addition to those previously reported, somata with SPLI were found in the dorsal lateral posterior groups #1 and #2, the ventral lateral posterior groups #1 and #2, and several distinct subgroups of the medial and ventral medial groups. In the retina of the lateral eye, fibers with both FLI and SPLI ramify in the lateral plexus and ultimately innervate the corneal epidermis. Brain homogenates were examined for immunoreactive (ir) FMRFamide and ir-substance P with radioimmunoassay techniques. Ir-FMRFamide and ir-substance P eluted in different fractions from both gel filtration chromatography and HPLC. Furthermore, the binding curves for both substances were similar to those of the corresponding synthetic compounds. Brain homogenates were also bioassayed on the lateral eye. Three gel filtration fractions mimic natural circadian activity by increasing the sensitivity of the lateral eye, but they were not coincident with ir-FMRFamide or ir-substance P. Although it is not completely resolved what the active molecules in these fractions are, it is clear that neither ir-FMRFamide nor ir-substance P is a possible candidate.

The Sequences of Five Neuropeptides Isolated from Limulus using Antisera to FMRFamide

“…Notwithstanding that FaRPs have been detected immunocytochemically in the lateral eye of the horseshoe crab (Lewandowski et al, 1989), we could detect no immunoreactive fractions in an extract of 50 lateral eyes.…”

“…In ad- Received 7 December 1992;accepted 29 March 1993. dition, efferent axons that project to the lateral eyes of Limulus also contain FMRFamide-like immunoreactivity (Lewandowski et al, 1989), though these fibers seem to be part of a general epidermal innervation rather than being restricted to the ommatidia. In any event, none of the peptides responsible for this immunoreactivity in Limulus have been sequenced.…”

Circadian efferent input to Limulus eyes: Anatomy, circuitry, and impact

“…But these SP/FMRFamide efferents are part of a general cuticular innervation and entirely separate from the octopaminergic system. The axons do not penetrate lateral eye ommatidia (Lewandowski et al, 1989), and they are not present in the ventral optic nerve. They arise from cell clusters in the circumesophageal ring that project out the third and fourth dorsal segmental nerves, and they join the lateral optic nerve near the lateral eye.…”

Dual controls for screening pigment movement in photoreceptors of theLimuluslateral eye: Circadian efferent input and light

“…Battelle and Kass and their collaborators have built a strong case for octopamine as one of the efferent neurotransmitters (Battelle, 1980;Battelle et al, 1982;Evans et al, 1983;Battelle & Evans, 1984;Kass & Barlow, 1984;Kass et al, 1988)-one which may act through a cAMP second messenger system (Kaupp et al, 1982;Edwards & Battelle, 1987;Kass et al, 1988). However, there is evidence that other substances are released from the efferent terminals -one of which may be an octopamine conjugate (Battelle & Evans, 1986;Battelle et al, 1988) and one of which may be an unidentified neuropeptide (Lehman & Barlow, 1987a,i>) that is not substance P (Lehman et al, 1988;Lewandowski et al, 1989). Further examination of possible roles of these putative neurotransmitters in triggering pigment migration is warranted.…”