Calcium-bearing objects elicit shell selection behavior in a hermit crab.

Hermit crabs explore empty gastropod shells by touching, rolling, and probing them before choosing one for a home. This component of shell selection behavior was examined in Pagurus hirsutiusculus hirsutiusculus (Dana) with binary choice tests between natural shells and accurate replicas of the shells with different chemical compositions. The results show that calcium emanating from the surface of shells is responsible for the behavior. Sensitivity to calcium may be a factor that enables the hermit crab to locate partially buried shells and discriminate empty shells from ones housing living gastropods or from small pebbles.

Dopamine-synthesizing neurons include the putative H-cell homologue in the moth Manduca sexta.

The catecholamine dopamine (DA) plays a fundamental role in the regulation of behavior and neurodevelopment across animal species. Uncovering the embryonic origins of neurons that express DA opens a path for a deeper understanding of how DA expression is regulated and, in turn, how DA regulates the activities of the nervous system. In a well-established insect model, Manduca sexta, we identified the putative homologue of the embryonic grasshopper "H-cell" using intracellular techniques, laser scanning confocal microscopy, and immunohistochemistry. In both species, this neuron possesses four axons and has central projections resembling the letter H. The H-cell in grasshoppers is known to be derived from the midline precursor 3 cell (MP3) and to pioneer the pathways of the longitudinal connectives; in Drosophila, the H-cell is also known to be derived from MP3. In the current study, we demonstrate that the Manduca H-cell is immunoreactive to antibodies raised against DA and its rate-limiting synthetic enzyme, tyrosine hydroxylase (TH). In larvae and adults, one DA/TH-immunoreactive (-ir) H-cell per ganglion is present. In embryos, individual ganglia contain a single midline TH-ir cell body positioned along side its putative sibling. Such observations are consistent with the known secondary transformation (in grasshoppers) of only one of the two MP3 progeny during early development. Although a hallmark feature of invertebrate neurons is the fairly stereotypical position of neuronal somata, we found that the H-cell somata can "flip-flop" by 180 degrees between an anterior and posterior position. This variability appears to be random and is not restricted to any particular ganglion. Curiously, what is segment-specific is the absence of the DA/TH-ir H-cell in the metathoracic (T3) ganglion as well as the unique structure of the H-cell in the subesophageal ganglion. Because this is the first immunohistochemical study of DA neurons in Manduca, we have provided the distribution pattern and morphologies of dopaminergic neurons, in addition to the H-cells, within the ventral nerve cord during development.

Steroid regulation of octopamine expression during metamorphic development of the moth Manduca sexta.

Octopamine (OA), a biogenic amine similar to norepinephrine, has profound and well-documented actions on the nervous systems of invertebrates. In the insect, Manduca sexta, we examined the developmental plasticity of OA synthesis, studied its endocrine regulation, and observed previously undescribed OA-immunoreactive (ir) neurons. We found that levels of tyramine beta-hydroxylase (TbetaH), an essential enzyme for the biosynthesis of OA, increase during metamorphosis. Based on the established and influential roles of the steroid hormone 20-hydroxyecdysone (20-HE) during development, we tested the hypothesis that increases in TbetaH levels and OA immunoreactivity are regulated by the rise in 20-HE occurring during pupal-adult development. We determined that the levels of TbetaH in the terminal abdominal ganglion (neuromeres 6-9) remain at a constant level during pupal development and the early stages of adult development. Beginning at ca. pupal stage 8, however, the levels of TbetaH begin to rise, reaching a maximum level by pupal stage 12. By removing the source of ecdysteroid hormone through ligation, and by subsequent replacement of 20-HE via infusion, we found evidence indicating that the preadult rise of 20-HE is both necessary and sufficient for the increased levels of TbetaH. During the course of our study, we also identified previously unreported OA-ir neurons. In particular, adult-specific OA-ir lateral cells were found, as were relatively small OA-ir dorsal median pairs that doubled in size during adult development. Abdominal ganglia not exposed to the preadult rise in 20-HE possessed neither the OA-ir lateral neurons nor the somatic growth of the smaller OA-ir median neurons. These newly described OA-ir neurons probably contribute to the steroid-induced elevations of TbetaH observed at the end of metamorphosis.

Distribution and developmental expression of octopamine-immunoreactive neurons in the central nervous system of the leech.

Octopamine, a biogenic amine analogous to norepinephrine, plays an important role in the orchestration and modulation of invertebrate behavior. In the leech, the behavioral actions of octopamine have been demonstrated; however, identification of octopaminergic neurons had not been determined by using immunohistochemical techniques. Thus, we used an antibody highly specific to octopamine to examine the distribution of octopamine-immunoreactive neurons in the segmental ganglia of American and European medicinal leeches (Macrobdella decora and Hirudo medicinalis). One pair of octopamine-immunoreactive neurons was located in the dorsolateral ganglionic region of anterior ganglia 1-6 and posterior ganglia 15-21. No corresponding octopamine-immunoreactive neurons were found in midbody ganglia 7-14. Using Neutral Red staining in combination with intracellular Neurobiotin injections and octopamine immunostaining, we determined the identity of the dorsolateral octopamine-immunoreactive cells. The dorsolateral octopamine-immunoreactive neuron (the DLO) was not cell 21, the only previously reported Neutral Red staining neuron in the dorsolateral position. We also determined that the Leydig neuron was not octopamine immunoreactive in either of the two medicinal leech species. Octopamine immunostaining in the sex ganglia revealed hundreds of immunoreactive neurons in sexually mature leeches. Such neurons were not observed in juvenile leeches. The developmental time course of octopamine immunoreactivity in the dorsolateral octopamine-immunoreactive neurons was also investigated by staining embryonic Hirudo medicinalis. Octopamine expression occurred relatively late as compared with the detectable onset of serotonin expression. Octopamine expression in the dorsolateral octopamine-immunoreactive cells was not detectable at early to mid-embryonic stages, and must commence during late embryonic to early juvenile stages. The identification of octopamine-immunoreactive cells now sets the stage for further investigations into the functional role of octopamine in leech behavior and the development of behavior.

Novel mouse IgG-like immunoreactivity expressed by neurons in the moth Manduca sexta: developmental regulation and colocalization with crustacean cardioactive peptide.

Immunoglobulin-related molecules have been shown to play important roles in cell-cell recognition events during the development of both vertebrate and invertebrate nervous systems. In the moth, Manduca sexta, we report the presence of novel, mouse, immunoglobulin G (mIgG)-like immunoreactivity in a discrete population of identified neurosecretory neurons (the NS-Ls also known as the cell 27s) and interneurons (the IN-704s). A number of polyclonal anti-mIgG antibodies were used to immunostain these cells in wholemount. The mIgG-like-immunoreactive (IR) neurons were present during embryogenesis through the developing adult stages, but disappeared in the postemerged adult. Biochemical analysis of M. sexta ventral nerve cords revealed that the mIgG-like antigen is a membrane-associated 27-kDa protein which is likely responsible for the mIgG-like immunostaining observed. Unambiguous identification of the mIgG-like-IR neurons was based on neuronal morphology and our ability to demonstrate conclusively that these neurons expressed immunoreactivity to an antiserum against crustacean cardioactive peptide (CCAP). The NS-Ls and IN-704s were both shown to colocalize the CCAP and mIgG-like immunoreactivities. The mIgG-like and CCAP-IR neurons were identical to a subset of CCAP-IR neurons recently described by Davis et al. [(1993) J. Comp. Neurol., 338:612-627] in pupae. We found these CCAP-IR neurons, however, also to be present in larvae. The mIgG-like- and CCAP-IR neurons included the NS-L pair of the subesophageal maxillary neuromere, which projected anteriorly to the corpora cardiaca, and the NS-L of the labial neuromere whose axons projected out the dorsal nerve of the next posterior ganglion. The mIgG-like and CCAP-IR NS-Ls were also observed throughout the three thoracic ganglia, and all shared strikingly similar structural features. These cells exited out the dorsal nerve of the next posterior ganglion and eventually projected to the neurohemal release sites of the perivisceral organs. These neurons appear to be the homologues of the abdominal CCAP-IR NS-Ls, neurons that in the adult switch their neurotransmitter and release the neuropeptide bursicon. Our description of the distribution and developmental expression of this novel mIgG-like immunoreactivity may provide new insights into the regulation of neurotransmitter plasticity and/or recognition-signaling events involved in the embryonic and postembryonic assembly of the nervous system.

A light insensitive method for contrast enhancement of insect neurons filled with a cobalt-lysine complex.

Modified protocols for cobalt-filling and silver intensification of neurons in the larval and adult stages of the moth, Manduca sexta, have led to improved neuronal visualization and minimal background staining. In particular, long distance projecting multisegmental interneurons, originating in the pterothoracic or terminal abdominal ganglion, were best visualized when a cobalt:lysine complex was used to fill hemi-connectives for several days at 4 C. Ganglia closest to the placement of tracer, which became flooded with cobalt:lysine during the filling period, were removed from the insect. This step eliminated the artifactual filling of neurons that may have taken up the tracer from such pooled regions. This led to a more accurate assessment of whether a multisegmental interneuron projected through the full length of nerve cord to the original site of tracer placement. The protocol for light insensitive silver intensification of cobalt-filled neurons was modified to include an important pH adjustment. NaOH was used to alter the pH of the protective colloid, sodium tungstate, to 10.4 or greater in solution. Especially in larvae, our techniques produced intensely stained cobalt-filled neurons within ganglia that remained transparent and relatively free of nonspecific silver deposition.

Metamorphosis of the ecdysis motor pattern in the hawkmoth, Manduca sexta.

The hawkmoth, Manduca sexta, undergoes periodic molts during its growth and metamorphosis. At the end of each molt, the old cuticle is shed by means of a hormonally-activated ecdysis behavior. The pharate adult, however, must not only shed its old cuticle but also dig itself out from its underground pupation chamber. To accomplish this, the adult performs a series of abdominal retractions and extensions; the extensions are coupled with movements of the wing bases. This ecdysis motor pattern is distinct from the slowly progressing, anteriorly-directed, abdominal peristalses expressed by ecdysing larvae and pupae. We have found that the ability to produce the larval-like ecdysis pattern is retained in the adult. Although this behavior is not normally expressed by the adult, larval-like ecdysis could be unmasked when descending neuronal inputs, originating in the pterothoracic ganglion, were removed from the unfused abdominal ganglia. Transformation of the adult-specific ecdysis pattern to the larval-like pattern was accomplished by transecting the connectives between the pterothorax and the abdomen, or by reversibly blocking neuronal activity with a cold-block. A comparative analysis of the ecdysis motor patterns expressed by larvae and by isolated adult abdomens indicates that the two motor patterns are indistinguishable, suggesting that the larval ecdysis motor pattern is retained through metamorphosis. We speculate that its underlying neural circuitry is conserved through development and later modulated to produce the novel ecdysis pattern expressed in the adult stage.

Coactivation of putative octopamine- and serotonin-containing interneurons in the medicinal leech.

Possible interactions between octopamine-immunoreactive (IR) and serotonergic neurons in the CNS of the medicinal leech were investigated. Simultaneous intracellular recordings of serotonin-containing neurons (either the Retzius neuron or cell 21) and the dorsolateral octopamine-IR (DLO) neuron demonstrated that both sets of neurons are coactive at times. Depolarization of either serotonergic cell 21 or the Retzius neuron did not alter the membrane potential of the DLO. Similarly, depolarization of the DLO did not affect the serotonergic neurons examined. Because it was found that the DLO and either the serotonergic cell 21 or Retzius neuron were at times coactive, we looked for possible sources of common excitatory inputs. The centrally located pressure (P)- and touch (T)-sensitive mechanosensory neurons excited the DLOs through a polysynaptic pathway. Stimulation of nociceptive (N) mechanosensory neurons did not cause a measurable depolarization in the membrane potential of the DLO. Through simultaneous recordings of the DLO, cell 21, and a particular identified mechanosensory neuron, it was demonstrated that activity in the T or P cells can excite both serotonergic cell 21 and the octopamine-IR DLO. These findings indicate that, in many instances, both serotonin and octopamine, biogenic amines with neuromodulatory actions in many different invertebrates, may be released simultaneously in the leech.

To swim or not to swim: regional effects of serotonin, octopamine and amine mixtures in the medicinal leech.

Focally treating the head brain of the medicinal leech Hirudo medicinalis with various biogenic amines affected the initiation, termination and maintenance of fictive swimming (i.e., the neural correlate of swimming). Application of serotonin to saline surrounding only the head brain inhibited fictive swimming, whereas removing serotonin induced swimming. This contrasts sharply with previous observations that serotonin applied to the nerve cord induces swimming. Although application of octopamine to the brain activated swimming, a mixture of octopamine and serotonin inhibited swimming. Subsequent removal of this mixture from the brain activated robust swimming and was more potent for activating swimming than either the removal of serotonin or the application of octopamine. Swim episodes induced by brain-specific manipulations of octopamine had more swim bursts per episode than those induced by serotonin. These brain-specific effects of the amines on fictive swimming are probably due to the modulation of higher-order circuits that control locomotion in the leech. We observed that serotonin or a mixture of serotonin and octopamine hyperpolarized an identified descending brain interneuron known as Tr2. Removal of the mixture caused Tr2 to exhibit membrane potential depolarizations that correlated in time with the expression of swim episodes.

Programmed cell death of an identified motoneuron examined in vivo: electrophysiological and morphological correlates.

A paucity of information exists about the electrophysiological and anatomical correlates of neurons committed to die in vivo. Thus, we examined how an identified neuron, motoneuron MN-12, dies during development in the intact moth Manduca sexta. The developmental programmed cell death of this motoneuron was examined because of its defined commitment point of death. In addition, its ability to be unambiguously identified between animals and its accessibility to recording and dye injection facilitated our examination. MN-12 becomes committed to die approximately 28-30 h after adult emergence. At this time, MN-12 can no longer be saved by manipulations of steroid hormone levels, protein synthesis, or removal of descending inputs. Our initial prediction was that within a few hours after the commitment point, MN-12 would begin showing a gradual loss of central arbors and alterations in membrane properties. Contrary to our expectations, we found the MN-12 motoneuron to exhibit a stable central morphology and electrophysiological profile for approximately 12 h, followed by a rapid dismantling that occurred within a 1- to 2-h period. Several hours prior to the commitment point, the target muscle of MN-12 was no longer viable; yet, this did not affect the death of MN-12 or cause retraction of its motor terminals. We conclude that the delayed (12-h) onset of rapid cell death is not preceded by a slow accumulation of damages to the neuronal membrane (e.g., ion channels or cytoskeletal components) as both the electrical activity and morphology of MN-12 remained measurably unaltered during this 12-h lag.

Improvements for the anatomical characterization of insect neurons in whole mount: the use of cyanine-derived fluorophores and laser scanning confocal microscopy.

The optical sectioning capability of the laser scanning confocal microscope was utilized to image dye-filled neurons within whole-mounted insect ganglia. Specific pterothoracic interneurons, in the moth Manduca sexta, were retrogradely filled with Neurobiotin and subsequently visualized with a monoclonal anti-biotin conjugated with one of the following fluorophores: fluorescein, and the newly developed cyanines, Cy3.18 (Cy3) and Cy5.18 (Cy5). Overall, the Cy5 fluorophore was best suited for imaging insect neurons within ganglia. This new methodology allowed us to identify and characterize morphologically a collection of descending multisegmental interneurons with large or small diameter somata. A variety of larger molecular weight (10,000 daltons) tracers was also used to examine the possibility of nonselective filling of neurons with Neurobiotin, possibly through gap junctions. We also investigated the usefulness of Cy3 and Cy5 as fluorophores for transmitter immunostaining of neurons in whole mount. Neurons immunoreactive for serotonin and the neuropeptides, FMRFamide and SCPB, were imaged in the brain and the pterothoracic ganglion. The central projections of some of these immunoreactive neurons were imaged in their entirety.

Mixtures of octopamine and serotonin have nonadditive effects on the CNS of the medicinal leech.

It is well established that neural networks respond to a wide variety of modulatory substances by which they can become reconfigured, yet few studies have examined the effects of neurotransmitter mixtures on such networks. In a previous study of the medicinal leech using triple intracellular recordings, we found that stimulation of identified mechanosensory neurons activated both the serotonergic cell 21 (a swimgating neuron) and the dorsal lateral octopamine (DLO) cell. Because these findings suggested that serotonin (5-HT) and octopamine (OA) may be released together, we investigated the effects of 5-HT and OA mixtures on isolated nerve cords of Hirudo medicinalis (which contained both head and tail brains). Fifty micromolar OA, 50 microM 5-HT, or a mixture of 50 microM OA and 50 microM 5-HT was bath applied to the nerve cord under constant perfusion conditions. Additional experiments were performed with combinations of either 25 or 100 microM OA and 5-HT. Neural activity was examined specifically in the segmentally repeated dorsal posterior (DP) nerve because it has been shown to contain identified swim motor units. Nonadditive effects of amine combinations were most apparent in their ability to decrease overall activity in the DP nerve and to alter patterned motor activity in the form of fictive swimming. Whereas swim burst activity has been previously shown to increase in nerve cords bathed in either 5-HT or OA solutions alone, we demonstrated that a mixture of the two amines resulted in a robust decrease in the number of swim bursts expressed and an inhibition of swim activity in preparations already swimming. Most compelling was the observation that when the amine mixture was replaced with normal saline, swim burst activity increased dramatically. We discuss that the effects of amine mixtures may be due to their interaction with descending interneurons known to trigger and inhibit swimming as the mixture-induced effects were not observed in nerve cords lacking the head and tail brains. Because the net effect of the two amines was not simply additive (i.e., 5-HT or OA is known to activate swimming, yet the mix inhibits swimming), this result reveals yet another layer of complexity inherent in "simpler" invertebrate nervous systems.

A motoneuron spared from steroid-activated developmental death by removal of descending neural inputs exhibits stable electrophysiological properties and morphology.

Neurons die during the development of nervous systems. The death of specific, identified motoneurons during metamorphosis of the tobacco hornworm, Manduca sexta, provides an accessible model system in which to study the regulation of postembryonic neuronal death. Hormones and descending neural inputs have been shown to influence the survival of abdominal motoneurons during the first few days of adult life in this insect. Motoneurons prevented from undergoing the normal process of developmental degeneration by removal of neural inputs were examined at the physiological and structural levels using several cell imaging techniques. Although these neurons lost their muscle targets and experienced the endocrine cue that normally triggers death, they showed no overt electrophysiological or morphological signs of degeneration. Thus, by appropriate intervention, the MN-12 motoneuron can be spared from developmental neuronal death and remain as a functioning supernumerary element in the mature nervous system.

Programmed cell death of identified peptidergic neurons involved in ecdysis behavior in the Moth, Manduca sexta.

The eclosion of the adult Manduca sexta moth is followed by a wave of cell death that eliminates up to 50% of the neurons of the central nervous system within the first few days of imaginal life. While the identity of some of the dying motoneurons has been established, that of most doomed neurons is unknown. Here, we show that the dying cells include peptidergic neurons involved in the control of ecdysis behavior. These cells belong to a small population of 50 neurons that express crustacean cardioactive peptide (CCAP), a potent regulator of the ecdysis motor program, and show increases in cyclic 3',5'-guanosine monophosphate at each ecdysis. First, we describe new markers for these neurons and show that they are expressed in these CCAP-immunoreactive neurons in a complex temporal pattern during development. We then show that these neurons die within 36 h after adult eclosion, the last performance of ecdysis behavior in the life of the animal, via the active, genetically determined process of programmed cell death. The death of these neurons supports the hypothesis that outmoded or unused neurons are actively eliminated.