Roger P. Croll

Development of catecholaminergic neurons in the pond snail, Lymnaea stagnalis: I. Embryonic development of dopamine-containing neurons and dopamine-dependent behaviors

The embryonic development of the catecholaminergic system of the pond snail, Lymnaea stagnalis, was investigated by using chromatographic and histochemical methods. High performance liquid chromatography suggested that dopamine was the only catecholamine present in significant concentrations throughout the embryonic development of Lymnaea. Dopamine first became detectable at about embryonic stage (E) 15 (15% of embryonic development) and then increased in amount during early development to reach about 120–140 fmol per animal by around E40. Dopamine content remained stable during mid‐embryogenesis (E40–65), increased slowing for the next couple of days, and then increased rapidly to culminate at about 400 fmol per animal by hatching. The detection of aldehyde‐ and glyoxylate‐induced fluorescence and of tyrosine hydroxylaselike immunoreactivity indicated that the first catecholaminergic cells appeared in the late trochophore or early veliger stage of embryonic development (E32–35). The paired perikarya of these transient apical catecholaminergic (TAC) neurons were located beneath the apical plate, remained outside of the central ganglia during embryogenesis, and no longer contained detectable catecholamines close to hatching. TAC neurons bore cilia on the ends of short processes that penetrated the overlying epithelium; their long processes branched repeatedly under the ciliated apical plate. Several smaller catecholaminergic cells first appeared in the anterior margin of the foot at a stage when the embryos began to metamorphose from the veliger form (E55). Similar bipolar cells later appeared in the tentacle and lips. The axons of all of these small peripheral cells projected centrally and terminated within the neuropil of different central ganglia. Central catecholaminergic neurons, including RPeD1, differentiated only after metamorphosis was complete (E75). Development of locomotor, respiratory, and feeding behaviors correlated with maturation of catecholaminergic neurons, as indicated by histology and chromatography. J. Comp. Neurol. 404:285–296, 1999.

Development of the larval nervous system of the gastropod Ilyanassa obsoleta

Gastropods have been well studied in terms of early cell cleavage patterns and the neural basis of adult behaviors; however, much less is known about neural development in this taxon. Here we reveal a relatively sophisticated larval nervous system in a well‐studied gastropod, Ilyanassa obsoleta. The present study employed immunocytochemical and histofluorescent techniques combined with confocal microscopy to examine the development of cells containing monoamines (serotonin and catecholamine), neuropeptides (FMRFamide and leu‐enkephalin related peptides), and a substance(s) reactive to antibodies raised against dopamine beta‐hydroxylase. Neurons were first observed in the apical organ and posterior regions during the embryonic trochophore stage. During later embryonic development neurons appeared in peripheral regions such as the foot, velum, and mantle and in the developing ganglia destined to become the adult central nervous system. In subsequent free‐swimming veliger stages the larval nervous system became increasingly elaborate and by late larval stages there existed ∼26–28 apical cells, 80–100 neurons in the central ganglia, and 200–300 peripherally located neurons. During metamorphosis some populations of neurons in the apical organ and in the periphery disappeared, while others were incorporated into the juvenile nervous system. Comparisons of neural elements in other molluscan larvae reveal several similarities such as comparable arrangements of cells in the apical organ and patterns of peripheral cells. This investigation reveals the most extensive larval nervous system described in any mollusc to date and information from this study will be useful for future experimental studies determining the role of larval neurons and investigations of the cellular and molecular mechanisms governing neural development in this taxon. J. Comp. Neurol. 466:197–218, 2003.

Histochemical localization of FMRFamide, serotonin and catecholamines in embryonic Crepidula fornicata (Gastropoda, Prosobranchia)

Abstract Recent reports indicate that neuronal elements develop in early larval stages of some Gastropoda from the Pulmonata and Opisthobranchia prior to the appearance of any ganglia of the future adult central nervous system (CNS). The present study describes similar early neuronal elements in Crepidula fornicata. A posterior FMRFamide-like immunoreactive (LIR) cell with anteriorly projected fibers was observed in the trochophore stage. Additional FMRFamide-LIR and serotonin-LIR cells and fibers were found in the apical organ in the trochophore and early veliger stages. FMRFamide-LIR and serotonin-LIR projections to the velum and foot were also detected at this time. As the veliger developed, peripheral FMRFamide-LIR and later catecholaminergic cells were located in the foot region. Also during this stage, catecholaminergic cells and processes were observed near the mouth. In addition, this study tentatively identified the first serotonin- and FMRFamide-LIR cells and fibers within the developing ganglia of the adult CNS, which appeared in close proximity to the earlier developing elements. These observations are consistent with the hypothesis that, in addition to its presumed role in the control of larval behaviors, the larval nervous system guides the development of the adult CNS. Larvae from the class Bivalvia and other invertebrate phyla also have neuronal elements marked by the presence of FMRFamide, serotonin, and catecholamines, and, therefore, this study may provide additional insights into phylogenetic relationships of the Gastropoda with other representatives of the Mollusca and different invertebrate phyla.

Development of catecholaminergic neurons in the pond snail, Lymnaea stagnalis: II. Postembryonic development of central and peripheral cells

Catecholamines have long been thought to play important roles in different mollusc neural functions. The present study used glyoxylate‐ and aldehyde‐induced histofluorescence to identify central and peripheral catecholaminergic neurons in the snail Lymnaea stagnalis. The majority of these cells were also found to react to antibodies raised against tyrosine hydroxylase. A minority of the catecholaminergic neurons, however, exhibited no such immunoreactivity. The number of central catecholaminergic neurons nearly doubled (from about 45 to about 80 cells) during the first 2–3 days of postembryonic development. Thereafter, catecholaminergic neurons again doubled in number and generally grew by about 100–200% in soma diameter as the snails grew by 1,000% in overall linear measurements. In contrast to the relatively meager addition of central catecholaminergic neurons, several thousand catecholaminergic somata were added to different peripheral tissues during postembryonic development. These small, centrally projecting neurons were particularly concentrated in the lips, esophagus, anterior margin of the foot, and different regions of the male and female reproductive tracts. Chromatographic analyses indicated that dopamine was the major catecholamine present in the central ganglia, foot, and esophagus, although detectable levels of norepinephrine (approximately 20% of dopamine levels) were also found in the ganglia. The total content but not the concentration of dopamine increased within the tissue samples during postembryonic development. The companion study (Voronezhskaya et al. [1999] J. Comp. Neurol. 404:285–296) and the present study furnish a complete description of central and peripheral catecholaminergic neurons from their first appearance in early embryonic development to adulthood. J. Comp. Neurol. 404:297–309, 1999.

Olfactory conditioning in the zebrafish (Danio rerio)

The zebrafish olfactory system is an attractive model for studying neural processing of chemosensory information. Here we characterize zebrafish olfactory behaviors and their modification through learning, using an apparatus consisting of a circular flow-through tank that allows controlled administration of odorants. When exposed to the amino acids l-alanine and l-valine, naive zebrafish responded with appetitive swimming behavior, which we measured as the number of >90 degrees turns made during 30s observation periods. Such appetitive responses were not observed when naive zebrafish were exposed to an unnatural odorant, phenylethyl alcohol (PEA). Repeated pairing of amino acids or PEA (conditioned stimuli, CS) with food flakes (unconditioned stimuli; UCS) increased odorant-evoked appetitive swimming behavior in all fish tested. The zebrafish also learned to restrict this behavior to the vicinity of a feeding ring, through which UCS were administered. When both nares were temporarily occluded, conditioned fish failed to respond to odorants, confirming that these behaviors were mediated by olfaction. These results represent the first demonstration of a classically conditioned appetitive response to a behaviorally neutral odorant in fish. Furthermore, they complement recent demonstrations of conditional place preferences in fish. By virtue of its robustness and simplicity, this method will be a useful tool for future research into the biological basis of olfactory learning in zebrafish.

Catecholamines modulate metamorphosis in the opisthobranch gastropod Phestilla sibogae.

Larvae of the nudibranch Phestilla sibogae are induced to metamorphose by a factor from their adult prey, the coral Porites compressa. Levels of endogenous catecholamines increase 6 to 9 days after fertilization, when larvae become competent for metamorphosis. Six- to nine-day larvae, treated with the catecholamine precursor L-DOPA (0.01 mM for 0.5 h), were assayed for metamorphosis in response to coral inducer and for catecholamine content by high-performance liquid chromatography. L-DOPA treatment caused 20- to 50-fold increases in dopamine, with proportionally greater increases in younger larvae, so that L-DOPA-treated larvae of all ages contained similar levels of dopamine. A much smaller (about twofold) increase in norepinephrine occurred in all larvae. The treatment significantly potentiated the frequency of metamorphosis of 7- to 9-d larvae at low concentrations of inducer. In addition, L-DOPA treatment at 9 d increased aldehyde-induced fluorescence in cells that were also labeled in the controls, and revealed additional cells. However, all labeled cells were consistent with the locations of cells showing tyrosine-hydroxylase-like immunoreactivity. Catecholamines are likely to modulate metamorphosis in P. sibogae, but rising levels of catecholamines around the time of competence are insufficient alone to account for sensitivity to inducer in competent larvae.

Development of Embryonic Cells Containing Serotonin, Catecholamines, and FMRFamide-Related Peptides in Aplysia californica

This study demonstrates the presence of a relatively extensive but previously unrecognized nervous system in embryonic stages of the opisthobranch mollusc Aplysia californica. During the trochophore stage, two pairs of cells were observed to be reactive to antibodies raised against the neuropeptides FMRFamide and EFLRIamide. These cells were located in the posterior region of the embryo, and their anterior projections terminated under the apical tuft. As the embryos developed into veliger stages, serotonin-like immunoreactive (LIR) cells appeared in the apical organ and were later observed to innervate the velum. Also, aldehyde-induced fluorescence indicative of catecholamines was present in cells in the foot, oral, and possibly apical regions during late embryonic veliger stages. Just before the embryo hatches as a free-swimming veliger, additional FMRFamide-LIR and catecholamine-containing cells appeared in regions that correspond to the ganglia of what will become the adult central nervous system (CNS). Neurons and connectives that will contribute to the adult CNS appear to develop along the pathways that are pioneered by the earliest posterior FMRFamide-LIR cells. These observations are consistent with the hypothesis that, besides their presumed roles in the control of embryonic behaviors, some elements may also guide the development of the CNS. Embryonic nervous systems that develop prior to and outside of the adult CNS have also been reported in pulmonate and prosobranch species of molluscs. Therefore, the demonstration of early developing neurons and their transmitter phenotypes in A. californica presents new opportunities for a better understanding of the ontogeny and phylogeny of both behavioral and neuronal function in this important model species.

Structure and autonomic innervation of the swim bladder in the zebrafish (Danio rerio)

Many teleosts actively regulate buoyancy by using a gas‐filled swim bladder, which is thought to be under autonomic control. Here we investigated the swim bladder in the zebrafish to determine possible mechanisms of gas‐content regulation. Fluorescently labelled phalloidin revealed myocytes that appeared to form a possible sphincter at the junction of the pneumatic duct and esophagus. Myocytes also formed thick bands along the ventral surface of the anterior chamber and bilaterally along the posterior chamber. Thinner layers of myocytes were located elsewhere. Staining of peroxidase within erythrocytes revealed a putative rete and smaller blood vessels in muscle bands and elsewhere. The antibodies zn‐12, a general neuronal marker, and SV2, a synaptic vesicle marker labelling presynaptic terminals, revealed widespread innervation of the swim bladder system. Widespread innervation of the swim bladder was also indicated by acetylcholinesterase histochemistry, but choline acetyltransferase‐immunoreactive (‐IR) somata and fibers were limited to the junction of the pneumatic duct and esophagus. In contrast, varicose tyrosine hydroxylase‐IR fibers innervated muscles and blood vessels throughout the system. Neuropeptide Y‐IR somata were located near the junction of the duct and esophagus and varicose fibers innervated muscles and vasculature of the posterior chamber and duct. Vasoactive intestinal polypeptide immunoreactivity was abundant throughout the anterior chamber but sparsely distributed elsewhere. Serotonin‐IR fibers and varicosities were located only along blood vessels near the junction of the pneumatic duct and posterior chamber. Our results suggest that the zebrafish swim bladder is a complex and richly innervated organ and that buoyancy‐regulating effectors may be controlled by multiple populations of autonomic neurons. J. Comp. Neurol. 495:587–606, 2006.

Neuronal development in larval mussel Mytilus trossulus (Mollusca: Bivalvia)

Although our understanding of neuronal development in Trochozoa has progressed substantially in recent years, relatively little attention has been paid to the bivalve molluscs in this regard. In the present study, the development of FMRFamide-, serotonin- and catecholamine-containing cells in the mussel, Mytilus trossulus, was examined using immunocytochemical and histofluorescent techniques. Neurogenesis starts during the trochophore stage at the apical extreme with the appearance of one FMRFamide-like immunoreactive (lir) and one serotonin-lir sensory cell. Later, five FMRFamide-lir and five serotonin-lir apical sensory cells appear, and their basal fibres form an apical neuropil. Fibres of two lateral FMRFamide-lir apical cells grow posteriorly and at the time that they reach the developing foot, the first FMRFamide-lir neurons of the pedal ganglia also appear. Subsequently, FMRFamide-lir fibres grow further posteriorly and reach the caudal region where neurons of the developing visceral ganglia then begin to appear. In contrast, the five apical serotonin-lir neurons do not appear to project outside the apical neuropil until the late veliger stage. Catecholamine-containing cells are first detected in the veliger stage where they appear above the oesophagus, and subsequently in the velum, foot, and posterior regions. Though neural development in M. trossulus partly resembles that of polyplacophorans in the appearance of the early FMRFamidergic elements, and of scaphopods in the appearance of the early serotonergic elements, the scenario of neural development in M. trossulus differs considerably from that of other Trochozoa (bivalves, gastropods, polyplacophorans, scaphopods and polychaetes) studied to date.

Identified Neurons and Cellular Homologies

Studies on homologies of identified neurons offer the promise for an understanding of the evolution of gross neural structures and behaviors in terms of the evolution of single nerve cells. Strong cases now exist in the literature for cellular homologies and evidence is available that permits an initial evaluation of which specific features of nerve cells appear to be most conserved through evolution and which features appear to be plastic and therefore permit adaptive variations in the morphology of the nervous system and in its behavioral manifestations. However, due to the relatively small number of putative cellular homologies which have been studied to date, generalizations may be of questionable accuracy. Much more information is necessary in the form of more examples of identifiable cells with known functions. Such examples will possibly allow better insights into how nerve cells adapt to pressures for changes in function. New techniques must also be employed which allow for the sampling of different types of cells than have usually been identified and homologized in the past. Finally, broader phyletic surveys of such neurons are also necessary to test the generality of hypotheses on the conservation and plasticity of neuronal features through evolution.