Transition from marine to terrestrial ecologies: changes in olfactory and tritocerebral neuropils in land-living isopods.

In addition to the ancestors of insects, representatives of five lineages of crustaceans have colonized land. Whereas insects have evolved sensilla that are specialized to allow the detection of airborne odors and have evolved olfactory sensory neurons that recognize specific airborne ligands, there is so far little evidence for aerial olfaction in terrestrial crustaceans. Here we ask the question whether terrestrial Isopoda have evolved the neuronal substrate for the problem of detecting far-field airborne chemicals. We show that conquest of land of Isopoda has been accompanied by a radical diminution of their first antennae and a concomitant loss of their deutocerebral olfactory lobes and olfactory computational networks. In terrestrial isopods, but not their marine cousins, tritocerebral neuropils serving the second antenna have evolved radical modifications. These include a complete loss of the malacostracan pattern of somatotopic representation, the evolution in some species of amorphous lobes and in others lobes equipped with microglomeruli, and yet in others the evolution of partitioned neuropils that suggest modality-specific segregation of second antenna inputs. Evidence suggests that Isopoda have evolved, and are in the process of evolving, several novel solutions to chemical perception on land and in air.

Conserved and convergent organization in the optic lobes of insects and isopods, with reference to other crustacean taxa.

The shared organization of three optic lobe neuropils-the lamina, medulla, and lobula-linked by chiasmata has been used to support arguments that insects and malacostracans are sister groups. However, in certain insects, the lobula is accompanied by a tectum-like fourth neuropil, the lobula plate, characterized by wide-field tangential neurons and linked to the medulla by uncrossed axons. The identification of a lobula plate in an isopod crustacean raises the question of whether the lobula plate of insects and isopods evolved convergently or are derived from a common ancestor. This question is here investigated by comparisons of insect and crustacean optic lobes. The basal branchiopod crustacean Triops has only two visual neuropils and no optic chiasma. This finding contrasts with the phyllocarid Nebalia pugettensis, a basal malacostracan whose lamina is linked by a chiasma to a medulla that is linked by a second chiasma to a retinotopic outswelling of the lateral protocerebrum, called the protolobula. In Nebalia, uncrossed axons from the medulla supply a minute fourth optic neuropil. Eumalacostracan crustaceans also possess two deep neuropils, one receiving crossed axons, the other uncrossed axons. However, in primitive insects, there is no separate fourth optic neuropil. Malacostracans and insects also differ in that the insect medulla comprises two nested neuropils separated by a layer of axons, called the Cuccati bundle. Comparisons suggest that neuroarchitectures of the lamina and medulla distal to the Cuccati bundle are equivalent to the eumalacostracan lamina and entire medulla. The occurrence of a second optic chiasma and protolobula are suggested to be synapomorphic for a malacostracan/insect clade.

Development of laminar organization in the mushroom bodies of the cockroach: Kenyon cell proliferation, outgrowth, and maturation.

The mushroom bodies of the insect brain are lobed integration centers made up of tens of thousands of parallel-projecting axons of intrinsic (Kenyon) cells. Most of the axons in the medial and vertical lobes of adult cockroach mushroom bodies derive from class I Kenyon cells and are organized into regular, alternating pairs (doublets) of pale and dark laminae. Organization of Kenyon cell axons into the adult pattern of laminae occurs gradually over the course of nymphal development. Newly hatched nymphs possess tiny mushroom bodies with lobes containing a posterior lamina of ingrowing axons, followed by a single doublet, which is flanked anteriorly by a gamma layer composed of class II Kenyon cells. Golgi impregnations show that throughout nymphal development, regardless of the number of doublets present, the most posterior lamina serves as the "ingrowth lamina" for axons of newborn Kenyon cells. Axons of the ingrowth lamina are taurine- and synaptotagmin-immunonegative. They produce fine growth cone tipped filaments and long perpendicularly oriented collaterals along their length. The maturation of these Kenyon cells and the formation of a new lamina are marked by the loss of filaments and collaterals, as well as the onset of taurine and synaptotagmin expression. Class I Kenyon cells thus show plasticity in both morphology and transmitter expression during development. In a hemimetabolous insect such as the cockroach, juvenile stages are morphologically and behaviorally similar to the adult. The mushroom bodies of these insects must be functional from hatching onward, while thousands of new neurons are added to the existing structure. The observed developmental plasticity may serve as a mechanism by which extensive postembryonic development of the mushroom bodies can occur without disrupting function. This contrasts with the more evolutionarily derived holometabolous insects, such as the honey bee and the fruit fly, in which nervous system development is accomplished in a behaviorally simple larval stage and a quiescent pupal stage.

Taurine-, aspartate- and glutamate-like immunoreactivity identifies chemically distinct subdivisions of Kenyon cells in the cockroach mushroom body.

The lobes of the mushroom bodies of the cockroach Periplaneta americana consist of longitudinal modules called laminae. These comprise repeating arrangements of Kenyon cell axons, which like their dendrites and perikarya have an affinity to one of three antisera: to taurine, aspartate, or glutamate. Taurine-immunopositive laminae alternate with immunonegative ones. Aspartate-immunopositive Kenyon cell axons are distributed across the lobes. However, smaller leaf-like ensembles of axons that reveal particularly high affinities to anti-aspartate are embedded within taurine-positive laminae and occur in the immunonegative laminae between them. Together, these arrangements reveal a complex architecture of repeating subunits whose different levels of immunoreactivity correspond to broader immunoreactive layers identified by sera against the neuromodulator FMRFamide. Throughout development and in the adult, the most posterior lamina is glutamate immunopositive. Its axons arise from the most recently born Kenyon cells that in the adult retain their juvenile character, sending a dense system of collaterals to the front of the lobes. Glutamate-positive processes intersect aspartate- and taurine-immunopositive laminae and are disposed such that they might play important roles in synaptogenesis or synapse modification. Glutamate immunoreactivity is not seen in older, mature axons, indicating that Kenyon cells show plasticity of neurotransmitter phenotype during development. Aspartate may be a universal transmitter substance throughout the lobes. High levels of taurine immunoreactivity occur in broad laminae containing the high concentrations of synaptic vesicles.

Learned discrimination of pattern orientation in walking flies.

To determine the pattern-orientation discrimination ability of blowflies, Phaenicia sericata, a learning/memory assay was developed in which sucrose served as the reward stimulus and was paired with one of two visual gratings of different orientations. Individual, freely walking flies with clipped wings were trained to discriminate between pairs of visual patterns presented in the vertical plane. During training trials, individual flies learned to search preferentially at the rewarded stimulus. In subsequent testing trials, flies continued to exhibit a learned preference for the previously rewarded stimulus, demonstrating an ability to discriminate between the two visual cues. Flies learned to discriminate between horizontal and vertical gratings, +45 degrees (relative to a 0 degrees vertical) and -45 degrees gratings, and vertical and +5 degrees gratings. Individual patterns of learning and locomotive behavior were observed in the pattern of exploration during training trials. The features of the visual cue critical for discrimination of orientation are discussed.

Exploitation of an ancient escape circuit by an avian predator: relationships between taxon-specific prey escape circuits and the sensitivity to visual cues from the predator.

The painted redstart Myioborus pictus uses visual displays to flush, pursue, and then capture an abundance of brachyceran Diptera that are equipped with giant fiber escape circuits. This paper investigates the relationships between features of the giant fiber system, the structure of visual stimuli produced by redstarts and their effectiveness in eliciting escape reactions by flies. The results show that dipterous taxa having large-diameter giant fibers extending short distances from the brain to motor neurons involved in escape are flushed at greater distances than taxa with longer and small-diameter giant fibers. The results of behavioral tests show the importance of angular acceleration of expanding image edges on the compound eye in eliciting escape responses. Lateral motion of stimulus profile edges as well as structured visual profiles additionally contribute to the sensitivity of one or more neural systems that trigger escape. Retinal subtense and angular velocity are known to trigger physiological responses in fly giant fiber circuits, but the contributions of edge length and lateral motion in a looming stimulus suggest that escape pathways might also receive inputs from circuits that are tuned to different types of motion. The present results suggest that these several properties of escape pathways have contributed to the evolution of foraging displays and plumage patterns in flush-pursuing birds.

Exploitation of an ancient escape circuit by an avian predator: prey sensitivity to model predator display in the field.

Certain insectivorous birds, such as the painted redstart (Myioborus pictus), undertake flush pursuit--a characteristic display that elicits an escape reaction by an insect, which the bird then chases in the air and eats. This account describes experiments showing that flush pursuit uses visual displays, which are likely to exploit an ancient neural circuit in dipteran insects, the visual systems of which are well documented as detecting looming stimuli and triggering an escape responses. Using models that decompose components of the redstart display, specific elements of the display were analyzed for their contribution in triggering visually induced escape behavior by dipterous insects. Elements tested were pivoting body movements, patterning on the spread tail and wings, and visual contrast of model redstarts against pale and dark backgrounds. We show that contrasting patterns within the plumage are crucial to foraging success, as is contrast of the bird against a background. Visual motion also significantly contributes to the successful flushing. In contrast, unpatterned models and patterned models that do not contrast with the background are less successful in eliciting escape responses of flies. Natural visual stimuli provided by Myioborus pictus are similar to those known to trigger looming and time-to-collision neurons in the escape circuits of flies and other insects, such as orthopterans. We propose that the tuning properties of these neural pathways might have contributed to the evolution of foraging displays in flush-pursuing birds.

Optic flow representation in the optic lobes of Diptera: modeling the role of T5 directional tuning properties.

An evolutionarily conserved system of small retinotopic neurons in dipteran insects, called bushy T-cells, provides information about directional motion to large collator neurons in the lobula plate. Physiological and anatomical features of these cells provide the basis for a model that is used to investigate requirements for generating optic flow selectivity in collators while allowing for evolutionary variations. This account focuses on the role of physiological tuning properties of T5 neurons. Various flow fields are defined as inputs to retinotopic arrays of T5 cells, the responses of which are mapped onto collators using innervation matrices that promote selectivity for flow type and position. Properties known or inferred from physiological and anatomical studies of neurons contributing to motion detection are incorporated into the model: broad tuning to local motion direction and the representation of each visual sampling unit by a quartet of small-field T5-like neurons with orthogonal preferred directions. The model predicts hitherto untested response properties of optic flow selective collators, and predicts that selectivity for a given flow field can be highly sensitive to perturbations in physiological properties of the motion detectors.

Optic flow representation in the optic lobes of Diptera: modeling innervation matrices onto collators and their evolutionary implications.

A network model of optic flow processing, based on physiological and anatomical features of motion-processing neurons, is used to investigate the role of small-field motion detectors emulating T5 cells in producing optic flow selective properties in wide-field collator neurons. The imposition of different connectivities can mimic variations observed in comparative studies of lobula plate architecture across the Diptera. The results identify two features that are crucial for optic flow selectivity: the broadness of the spatial patterns of synaptic connections from motion detectors to collators, and the relative contributions of excitatory and inhibitory synaptic outputs. If these two aspects of the innervation matrix are balanced appropriately, the network's sensitivity to perturbations in physiological properties of the small-field motion detectors is dramatically reduced, suggesting that sensory systems can evolve robust mechanisms that do not rely upon precise control of network parameters. These results also suggest that alternative lobula plate architectures observed in insects are consistent in allowing optic flow selective properties in wide-field neurons. The implications for the evolution of optic flow selective neurons are discussed.

Organization and significance of neurons that detect change of visual depth in the hawk moth Manduca sexta.

Visual stimuli representing looming or receding objects can be decomposed into four parameters: change in luminance; increase or decrease of area; increase or decrease of object perimeter length; and motion of the object's perimeter or edge. This paper describes intracellular recordings from visual neurons in the optic lobes of Manduca sexta that are selectively activated by certain of these parameters. Two classes of wide-field neurons have been identified that respond selectively to looming and receding stimuli. Class 1 cells respond to parameters of the image other than motion stimuli. They discriminate an approaching or receding disc from an outwardly or inwardly rotating spiral, being activated only by the disc and not by the spiral. Class 2 neurons respond to moving edges. They respond both to movement of the spiral and to an approaching or receding disc. These two classes are further subdivided into neurons that are excited by image expansion (looming) and are inhibited by image contraction (antilooming). Class 2 neurons also respond to horizontal and vertical movement of gratings over the retina. Stimulating class 1 and 2 neurons with white discs against a dark background results in the same activation as stimulation with dark discs against a white background, demonstrating that changes in luminance play no role in the detection of looming or antilooming. The present results show that the two types of looming-sensitive neurons in M. sexta use different mechanisms to detect the approach or retreat of an object. It is proposed that cardinal parameters for this are change of perimeter length detected by class 1 neurons and expansion or contraction visual flow fields detected by class 2 neurons. These two classes also differ with respect to their polarity, the former comprising centripetal cells from the optic lobes to the midbrain, the latter comprising centrifugal neurons from the midbrain to the optic lobes. The significance of these arrangements with respect to hovering flight is discussed.

Parallel organization in honey bee mushroom bodies by peptidergic Kenyon cells.

Antisera against the neuromodulatory peptides, Phe-Met-Arg-Phe-NH(2)-amide (FMRFamide) and gastrin cholecystokinin, demonstrate that the mushroom bodies of honey bees are subdivided longitudinally into strata. Three-dimensional reconstructions demonstrate that these strata project in parallel through the entire pedunculus and through the medial and vertical lobes. Immunostaining reveals clusters of immunoreactive cell bodies within the calyx cups and immunoreactive bundles of axons that line the inside of the calyx cup and lead to strata. Together, these features reveal that immunoreactive strata are composed of Kenyon cell axons rather than extrinsic elements, as suggested previously by some authors. Sorting amongst Kenyon cell axons into their appropriate strata already begins in the calyx before these axons enter the pedunculus. The three main concentric divisions of each calyx (the lip, collar, and basal ring) are divided further into immunoreactive and immunonegative zones. The lip neuropil is divided into two discrete zones, the collar neuropil is divided into five zones, and the basal ring neuropil is divided into four zones. Earlier studies proposed that the lip, collar, and basal ring are represented by three broad bands in the lobes: axons from adjacent Kenyon cell dendrites in the calyces are adjacent in the lobes even after their polar arrangements in the calyces have been transformed to rectilinear arrangements in the lobes. The universality of this arrangement is not supported by the present results. Although immunoreactive zones are found in all three calycal regions, immunoreactive strata in the lobes occur mainly in the two bands that were ascribed previously to the collar and the basal ring. In the lobes, immunoreactive strata are visited by the dendrites of efferent neurons that carry information from the mushroom bodies to other parts of the brain. Morphologically and chemically distinct subdivisions through the pedunculus and lobes of honey bees are comparable to longitudinal subdivisions demonstrated in the mushroom bodies of other insects, such as the cockroach Periplaneta americana. The functional and evolutionary significance of the results is discussed.

Olfactory systems: common design, uncommon origins?

In both vertebrates and invertebrates, odorant molecules reach the dendrites of olfactory receptor cells through an aqueous medium, which reflects the evolutionary origin of these systems in a marine environment. Important recent advances, however, have demonstrated striking interphyletic differences between the structure of vertebrate and invertebrate olfactory receptor proteins, as well as the organization of the genes encoding them. While these disparities support independent origins for odor-processing systems in craniates and protostomes (and even between the nasal and vomeronasal systems of craniates), olfactory neuropils share close neuroanatomical and physiological characters. Whereas there is a case to be made for homology among members of the two great protostome clades (the ecdysozoans and lophotrochozoans), the position of the craniates remains ambiguous.

Organization of olfactory and multimodal afferent neurons supplying the calyx and pedunculus of the cockroach mushroom bodies.

The mushroom bodies of neopteran insects are considered to be higher olfactory centers because their calyces receive abundant collaterals of projection neurons from the antennal lobes. However, intracellular recordings of mushroom body efferent neurons demonstrate that they respond to multimodal stimuli, implying that the mushroom bodies receive a variety of sensory cues. The present account describes new features of the organization of afferent neurons supplying the calyces of the cockroach Periplaneta americana. Afferent terminals segment the calyces into discrete zones, I, II, III, and IIIA, which receive afferents from 1) two discrete populations of sexually isomorphic olfactory glomeruli, 2) two types of male-specific olfactory glomeruli, 3) the optic lobes, and 4) multimodal interneurons that originate in protocerebral neuropils. In addition, intracellular recordings and dye fills show that at least four morphologically distinct GABAergic elements link many regions of the protocerebrum to the calyces. A new type of touch-sensitive centrifugal neuron has been identified terminating in the pedunculus. The dendrites of this afferent reside in satellite neuropil, beneath the mushroom body's medial lobe, which is supplied by collaterals from medial lobe efferent neurons and by terminals from the central complex. The role of this centrifugal cell in odorant sampling is considered. Golgi impregnation identifies other afferents in proximal regions of the calyx (zone IIIA) that also originate from satellite neuropils, suggesting major reafference from the medial lobes channeled through this region. The relevance of multimodal supply to the calyx in odorant discrimination is discussed as are comparisons between mushroom body organization in this phylogenetically basal neopteran and other taxa.

Representation of the calyces in the medial and vertical lobes of cockroach mushroom bodies.

Previous studies of honey bee and cockroach mushroom bodies have proposed that afferent terminals and intrinsic neurons (Kenyon cells) in the calyces are arranged according to polar coordinates. It has been suggested that there is a transformation by Kenyon cell axons of the polar arrangements of their dendrites in the calyces to laminar arrangements of their terminals in the lobes. Findings presented here show that cellular organization in the calyx of an evolutionarily basal neopteran, Periplaneta americana, is instead rectilinear, as it is in the lobes. It is shown that each calyx is divided into two halves (hemicalyces), each supplied by its own set of Kenyon cells. Each calyx is separately represented in the medial lobe where the dendritic trees of some efferent neurons receive inputs from one calyx only. Kenyon cell dendrites are arranged as narrow elongated fields, organized as rows in each hemicalyx. Dendritic fields arise from 14 to 16 sheets of Kenyon cell axons stacked on top of each other lining the inner surface of the calyx cup. A sheet consists of approximately 60 small bundles, each containing 5-15 axons that converge from the rim of the calyx to its neck. Each sheet contributes to a pair oflaminae, one dark one pale, called a doublet, that extends through the mushroom body. Dark laminae contain Kenyon cell axons packed with synaptic vesicles. Axons in pale laminae are sparsely equipped with vesicles. By analogy with photoreceptors, and with reference to field potential recordings, it is speculated that dark laminae are continuously active, being modulated by odor stimuli, whereas pale laminae are intermittently activated. Timm's silver staining and immunocytology reveal a second type of longitudinal division of the lobes. Five layers extend through the pedunculus and lobes, each composed of subsets of doublets. Four layers represent zones of afferent endings in the calyces. A fifth (the y layer) represents a specific type of Kenyon cell. It is concluded that the mushroom bodies comprise two independent modular systems, doublets and layers. Developmental studies show that new doublets are added at each instar to layers that are already present early in second instar nymphs. There are profound similarities between the mushroom bodies of Periplaneta, an evolutionarily basal taxon, and those of Drosophila melanogaster and the honey bee.

Multimodal efferent and recurrent neurons in the medial lobes of cockroach mushroom bodies.

Previous electrophysiological studies of cockroach mushroom bodies demonstrated the sensitivity of efferent neurons to multimodal stimuli. The present account describes the morphology and physiology of several types of efferent neurons with dendrites in the medial lobes. In general, efferent neurons respond to a variety of modalities in a context-specific manner, responding to specific combinations or specific sequences of multimodal stimuli. Efferent neurons that show endogenous activity have dendritic specializations that extend to laminae of Kenyon cell axons equipped with many synaptic vesicles, termed "dark" laminae. Efferent neurons that are active only during stimulation have dendritic specializations that branch mainly among Kenyon cell axons having few vesicles and forming the "pale" laminae. A new category of "recurrent" efferent neuron has been identified that provides feedback or feedforward connections between different parts of the mushroom body. Some of these neurons are immunopositive to antibodies raised against the inhibitory transmitter gamma-aminobutyric acid. Feedback pathways to the calyces arise from satellite neuropils adjacent to the medial lobes, which receive axon collaterals of efferent neurons. Efferent neurons are uniquely identifiable. Each morphological type occurs at the same location in the mushroom bodies of different individuals. Medial lobe efferent neurons terminate in the lateral protocerebrum among the endings of antennal lobe projection neurons. It is suggested that information about the sensory context of olfactory (or other) stimuli is relayed by efferent neurons to the lateral protocerebrum where it is integrated with information about odors relayed by antennal lobe projection neurons.

Mushroom bodies of the cockroach: their participation in place memory.

Insects and other arthropods use visual landmarks to remember the location of their nest, or its equivalent. However, so far, only olfactory learning and memory have been claimed to be mediated by any particular brain region, notably the mushroom bodies. Here we describe the results of experiments that demonstrate that the mushroom bodies of the cockroach (Periplaneta americana), already shown to be involved in multimodal sensory processing, play a crucial role in place memory. Behavioral tests, based on paradigms similar to those originally used to demonstrate place memory in rats, demonstrate a rapid improvement in the ability of individual cockroaches to locate a hidden target when its position is provided by distant visual cues. Bilateral lesions of selected areas of the mushroom bodies abolish this ability but leave unimpaired the ability to locate a visible target. The present results demonstrate that the integrity of the pedunculus and medial lobe of a single mushroom body is required for place memory. The results are comparable to the results obtained from hippocampal lesions in rats and are relevant to recent studies on the effects of ablations of Drosophila mushroom bodies on locomotion.

Mushroom bodies of the cockroach: activity and identities of neurons recorded in freely moving animals.

This article describes novel attributes of the mushroom bodies of cockroaches revealed by recording from neurons in freely moving insects. The results suggest several hitherto unrecognized functions of the mushroom bodies: extrinsic neurons that discriminate between imposed and self-generated sensory stimulation, extrinsic neurons that monitor motor actions, and a third class of extrinsic neurons that predict episodes of locomotion and modulate their activity depending on the turning direction. Electrophysiological units have been correlated with neurons that were partially stained by uptake of copper ions and silver intensification. Neurons so revealed correspond to Golgi-impregnated or Lucifer yellow-filled neurons and demonstrate that their processes generally ascend to other areas of the protocerebrum. The present results support the idea of multiple roles for the mushroom bodies. These include sensory discrimination, the integration of sensory perception with motor actions, and, as described in the companion article, a role in place memory.

Crustacean-insect relationships: the use of brain characters to derive phylogeny amongst segmented invertebrates.

Conserved neural characters identified in the brains of a variety of segmented invertebrates and outgroups have been used to reconstruct phylogenetic relationships. The analysis suggests that insects and crustaceans are sister groups and that the 'myriapods' are an artificial construct comprising unrelated chilopods and diplopods. Certain elements of the optic lobes and mid-brain support the notion that insects are more closely related to crustaceans than they are to any other arthropods. However, deep optic neuropils and optic chiasmata are homoplastic in insects and crustaceans. The organization of olfactory pathways suggests that insect olfactory lobes originated late, probably first appearing in orthopteroid or blattoid pterygotes. The present results are discussed with respect to recent studies on early development of arthropod nervous systems and the fossil record.

Functionally and anatomically segregated visual pathways in the lobula complex of a calliphorid fly.

In dipteran insects, the lobula plate neuropil provides a major efferent supply to the premotor descending neurons that control stabilized flight. The lobula plate itself is supplied by two major parallel retinotopic pathways from the medulla: small-field, magnocellular afferents that are implicated in achromatic motion processing and Y cells that connect the medulla with both the lobula plate and the lobula. A third pathway from the medulla involves transmedullary (Tm) neurons, which provide inputs to palisades of small-field neurons in the lobula. Although, in their passage to the brain, many output neurons from the lobula plate are separated physically from their counterparts in the lobula, there is an additional class of lobula complex output neurons. This group is composed of retinotopic lobula plate-lobula (LPL) and lobula-lobula plate (LLP) cells, each of which has dendrites in both the lobula and the lobula plate. The present account describes the anatomy and physiology of exemplars of LPL and LLP neurons, a wide-field tangential neuron that is intrinsic to the lobula complex, and representatives of the Tm- and Y-cell pathways. We demonstrate novel features of the lobula plate, which previously has been known as a motion-collating neuropil, and now also can be recognized as supporting direction- or nondirection-specific responses to local motion, encoding of contrast frequency, and processing of local structural features of the visual panorama.