Physiology and morphology of olfactory neurons associating with the protocerebral lobe of the honeybee brain

Physiology and morphology of olfactory neurons associated with the protocerebral lobe around the alpha-lobe of the mushroom body were studied in the brain of the honeybee Apis mellifera using intracellular recording and staining techniques. The responses of neurons to behaviorally relevant odorants (a blend, and components of the Nasonov pheromone, and some other non-pheromonal odors) were recorded. Different response patterns were observed within different neurons, and often within the same neuron, in response to different stimuli. All the neurons stained had innervations in the protocerebral lobe. The cell profiles varied from cells connecting the antennal lobe with both the protocerebral and lateral protocerebral lobes (projection neurons), cells linking the pedunculus of the mushroom body with both the protocerebral and lateral protocerebral lobes (PE1 neurons), cells linking the alpha-lobe and protocerebral lobe with the calyces of the mushroom body (feedback neurons), and cells linking the alpha-lobe and protocerebral lobe with the antennal lobe (recurrent neurons), to cells connecting the protocerebral lobe with the contralateral protocerebrum (bilateral neurons). These findings suggest that the protocerebral lobe acts as an olfactory center associating with other centers, and provides multi-layered recurrent networks within the protocerebrum and between the deutocerebrum and the protocerebrum in honeybee olfactory pathways.     

蟋蟀脑的解剖结构与生理机能

蟋蟀脑由前脑、中脑和后脑三部分组成。前脑由1对蕈形体、中央复合体和视叶构成;每个蕈形体由2个冠、柄及与柄相连的α叶和β叶组成,是信息联络整合部位;中央复合体由中央体和脑桥组成,主要参与感觉信息的加工过程;视叶由神经节层、外髓和内髓组成,是视觉系统的中心。中脑由主要组成成分为嗅觉纤维球的嗅叶组成,是嗅觉系统的中心。后脑向后与食道下神经节相连。     

Evolution of the central complex in the arthropod brain with respect to the visual system

Modular midline neuropils, termed arcuate body (Chelicerata, Onychophora) or central body (Myriapoda, Crustacea, Insecta), are a prominent feature of the arthropod brain. In insects and crayfish, the central body is connected to a second midline-spanning neuropil, the protocerebral bridge. Both structures are collectively termed central complex. While some investigators have assumed that central and arcuate bodies are homologous, others have questioned this view. Stimulated by recent evidence for a role of the central complex in polarization vision and object recognition, the architectures of midline neuropils and their associations with the visual system were compared across panarthropods. In chelicerates and onychophorans, second-order neuropils subserving the median eyes are associated with the arcuate body. The central complex of decapods and insects, instead, receives indirect input from the lateral (compound) eye visual system, and connections with median eye (ocellar) projections are present. Together with other characters these data are consistent with a common origin of arcuate bodies and central complexes from an ancestral modular midline neuropil but, depending on the choice of characters, the protocerebral bridge or the central body shows closer affinity with the arcuate body. A possible common role of midline neuropils in azimuth-dependent sensory and motor tasks is discussed.     

Locomotor control by the central complex in Drosophila-An analysis of the tay bridge mutant

Several aspects of locomotor control have been ascribed to the central complex of the insect brain; however, the role of distinct substructures of this complex is not well known. The tay bridge1 (tay1) mutant of Drosophila melanogaster was originally isolated on the basis of reduced walking speed and activity. In addition, tay1 is defective in the compensation of rotatory stimuli during walking and histologically, tay1 causes a mid-sagittal constriction of the protocerebral bridge, a constituent of the central complex. Cloning of the tay gene revealed that it encodes a novel protein with no significant homology to any known protein. To associate the behavioral phenotypes with the anatomical defect in the protocerebral bridge, we used different driver lines to express the tay cDNA in various neuronal subpopulations of the central brain in tay1-mutant flies. These experiments showed an association of the aberrant walking speed and activity with the structural defect in the protocerebral bridge. In contrast, the compensation of rotatory stimuli during walking was rescued without a restoration of the protocerebral bridge. The results of our differential rescue approach are supported by neuronal silencing experiments using conditional tetanus toxin expression in the same subset of neurons. These findings show for the first time that the walking speed and activity is controlled by different substructures of the central brain than the compensatory locomotion for rotatory stimuli.     

The projection of neuroendocrine fibers (NCC I and II) in the brains of three orthopteroid insects

Neuronal projections from neuroendocrine tracts (nervi corpori cordiaci I and II) in the brains of the locust (Schistocerca vaga), cricket (Acheta domesticus), and cockroach (Periplaneta americana) were studied using reconstructions of silver-intensified cobalt chloride preparations. Collaterals from the NCC I in these species branch extensively in the dorsal protocerebral neuropile, anterior to the stalk of the corpora pedunculata and ventral to its calyces. Other fibers project from the NCC I bilaterally into the medial protocerebral neuropile, anterior to the central body, and posterior to the beta lobes. NCC II collaterals arborize in the medial, dorsal, and lateral protocerebral neuropile, their region of projection partially overlapping with that of the NCC I. Several NCC II fibers terminate in the superior arch of the central body in Acheta but not in the other two species. Tritocerebral cells filled through the NCC I branch in the medial tritocerebral neuropile in all three species, but most extensively in Schistocerca. No NCC fibers were seen to penetrate any part of the corpora pedunculata, protocerebral bridge, olfactory glomeruli, ocellar tracts, or optic lobes. These neuronal projections from the NCC I and II lie anterior to regions of branching of second-order ocellar fibers and thus provide no anatomical basis for direct ocellar input to neurosecretory cells, contrary to previous reports for orthopteroid species (Brousse-Gaury, '71a, b). However, interneurons filled from the optic lobes were found to terminate in the same region of dorsal protocerebral neuropile as NCC I and II fibers in Acheta, thus providing a possible pathway for optic input to the cerebral neuroendocrine system.     

Immunocytochemical demonstration of octopamine-immunoreactive cells in the nervous system of Locusta migratoria and Schistocerca gregaria

Summary The distribution of octopamine in the metathoracic ganglion, brain and corpus cardiacum of Locusta migratoria and Schistocerca gregaria was investigated by means of immunocytochemistry with an antiserum against octopamine. The dorsal unpaired median (DUM) cells of the metathoracic ganglion were found to be strongly octopamine-immunoreactive. In the rostroventral part of the protocerebrum a group of seven immunopositive cells was demonstrated. Stained nerve fibres of these cells run into three directions: circumoesophageal connectives, midbrain, and optic lobes. As far as the protocerebrum is concerned, immunoreactive fibres were found in the central body, the protocerebral bridge, and in other neuropile areas. In the optic lobe a dense plexus of immunopositive fibres was found in the lobula and in the medulla. In the brain one other immunopositive cell was demonstrated, situated at the lateral border of the tritocerebrum. Octopamine could not be shown to occur either in the globuli cells of the mushroom bodies or in the dorsolateral part of the protocerebrum, where the perikarya of the secretomotor neurones are located that innervate the glandular cells of the corpus cardiacum. In the nervi corporis cardiaci II, which contain the axons of the neurones that extend into the glandular part of the corpus cardiacum, and in the corpus cardiacum proper no specific octopamine immunoreactivity could be found.     

Innervation pattern of suboesophageal ventral unpaired median neurones in the honeybee brain

In honeybees (Apis mellifera), the biogenic amine octopamine has been shown to play a role in associative and non-associative learning and in the division of labour in the hive. Immunohistochemical studies indicate that the ventral unpaired median (VUM) neurones in the suboesophageal ganglion (SOG) are putatively octopaminergic and therefore might be involved in the octopaminergic modulation of behaviour. In contrast to our knowledge about the behavioural effects of octopamine, only one neurone (VUMmx1) has been related to a behavioural effect (the reward function during olfactory learning). In this study, we have investigated suboesophageal VUM neurones with fluorescent dye-tracing techniques and intracellular recordings combined with intracellular staining. Ten different VUM neurones have been found including six VUM neurones innervating neuropile regions of the brain and the SOG exclusively (central VUM neurones) and four VUM neurones with axons in peripheral nerves (peripheral VUM neurones). The central VUM neurones innervate the antennal lobes, the protocerebral lobes (including the lateral horn) and the mushroom body calyces. Of these, a novel mandibular VUM neurone, VUMmd1, exhibits the same branching pattern in the brain as VUMmx1 and responds to sucrose and odours in a similar way. The peripheral VUM neurones innervate the antennal and the mandibular nerves. In addition, we describe one labial unpaired median neurone with a dorsal cell body, DUMlb1. The possible homology between the honeybee VUM neurones and the unpaired median neurones in other insects is discussed. This work was supported by the DFG ME 365/24-2.     

An electrophysiological study of olfactory interneurones in the brain of the honey-bee

Discharges of the olfactory units in the brain of the honey-bee were recorded extracellularly. Discharges were excitatory and inhibitory and showed on and on-off responses. In the on responses, phasic, phasic-tonic, and tonic patterns were found. No relation between discharge pattern and the region in the brain was found. The dominant pattern was phasic-tonic in the excitatory and tonic in the inhibitory units. Rebound or long-lasting phenomena seemed to be one of the characteristics of the neurones in the central nervous system.The latency of the response showed that olfactory information entering the deutocerebrum from the antenna was carried to the calyx of the ipsilateral brain via the antenno-cerebral tracts reported by Kenyon (1896), and then to the protocerebral lobe through the stalk of the mushroom body. The information was also carried to the contralateral brain after passing through the calyx of the ipsilateral brain or the central commissure.     

Physiological and anatomical properties of optical input-fibres to the mushroom body in the bee brain

More than 150 neurones in the nushroom body area of the bee brain were recorded and stained intracellularly with either Lucifer Yellow or Cobalt-Hexamminochloride (III). Among them 12 neurones have been characterized physiologically and anatomically which connect the medulla and the lobula with the mushroom bodies. All neurones responded to stationary or moving light stimuli exclusively. Movement-sensitive neurones were all direction-selective. Excitatory and inhibitory responses occurred in response to moving stripe patterns in the preferred and null directions respectively. Anatomically, the neurones could be clearly distinguished as belonging to three types depending on their input features in the optic lobes: (a) Neurones with small dendritic fields (up to 100 μm) in the lobula; (b) Neurones with large dendritic fields (up to 400 μm) in the lobula; (c) Neurones with small dendritic fields (up to 100 μm) in the medulla. The axons of all three cell types run from the optic lobes on each side to the outer ring tract around the pedunculus-calyx-transition and arborize in the collar region of the ipsilateral calyces. Additional branches invading the basal ring of the calyces had been observed; endings in the lip region were not found. The endings in the calyces often exhibited bleb-like specializations indicating their presynaptic nature. Retinotopic organization of the optic inputs into the calyces could not be proven. The results are compared with the characteristics of multimodal mushroom body output fibres and are discussed in context with the complex information processing and storage functions ascribed to the mushroom bodies.     

Evidence for the presence of serotonin in Mysidacea (Crustacea,Peracarida) as revealed by fluorescence immunohistochemistry

In crustaceans, serotonin (5-HT) exerts a wide range of physiological actions on many tissues. However, 5-HT has not been detected to date in Mysidacea (Crustacea, Peracarida). We have investigated the presence of 5-HT in the brain and the eyestalks of two Mysida (Leptomysis lingvura, Hemimysis margalefi) and one Lophogastrida (Lophogaster typicus) species by using the immunohistofluorescence technique. 5-HT-like immunopositive areas exhibit a similar pattern in the three species. 5-HT-like immunostaining is present in the retinular photosensitive cells, except in the deep-living species L. typicus. 5-HT-like cell bodies and fibres are observed in the lamina ganglionaris and in the three medullae. In the sinus gland, only 5-HT-like endings are detected. In the eyestalks, 5-HT-like fibres detected in the optic tract link with the protocerebrum, in which 5-HT-like somata and their extensions are found. Some neurones are detected in the anterior median cell cluster, in the protocerebral bridge and in the central body. In the deutocerebrum, the paracentral lobes are connected by immunoreactive fibres that run along the deutocerebral commissure. The glomeruli of the olfactory lobes exhibit strong diffuse immunostaining. Beside and in the median part of the deutocerebrum, at least two large serotoninergic neurones project their axons into the olfactory lobe cell cluster. Immunoreactive fibres are also found in the antennular neuropiles. Our results demonstrate the presence of 5-HT-like cell bodies and fibres in Mysidacea. The distribution patterns of the 5-HT immunoreactivity found herein are compared with those of other peracarids and decapods.     

Central complex substructures are required for the maintenance of locomotor activity in Drosophila melanogaster

In Drosophila melanogaster, former studies based on structural brain mutants have suggested that the central complex is a higher control center of locomotor behavior. Continuing this investigation we studied the effect of the central complex on the temporal structure of spontaneous locomotor activity in the time domain of a few hours. In an attempt to dissect the internal circuitry of the central complex we perturbed a putative local neuronal network connecting the four neuropil regions of the central complex, the protocerebral bridge, the fan-shape body, the noduli and the ellipsoid body. Two independent and non-invasive methods were applied: mutations affecting the neuroarchitecture of the protocerebral bridge, and the targeted expression of tetanus toxin in small subsets of central complex neurons using the binary enhancer trap P[GAL4] system. All groups of flies with a disturbed component of this network exhibited a common phenotype: a drastic decrease in locomotor activity. While locomotor activity was still clustered in bouts and these were initiated at the normal rate, their duration was reduced. This finding suggests that the bridge and some of its neural connections to the other neuropil regions of the central complex are required for the maintenance but not the initiation of walking. Accepted: 21 June 1999     

Neuronal pathways from the dorsal acelli of the house cricket,Acheta domesticus

Each ocellar nerve in the house cricket Acheta domesticus contains giant nerve fibers of 10-15 μ diameter, characterized in Golgi Cox preparations by a single row of short collaterals which runs along nearly the entire length of a fiber. Numerous long collaterals are given off by thin fibers in the ocellar nerve; medium-size fibers give off relatively few collaterals. The lateral ocellar tracts extend posteriorly through the dorsal protocerebrum, crossing the protocerebral bridge dorsally. The smaller median ocellar tract runs more ventrally through the pars intercerebralis; posterior to the bridge its fibers turn out toward the lateral nerves. Golgi and cobalt preparations reveal branching of giant and mediu_-size ocellar fibers posterior to the bridge at two levels, forming bilateral regions of ocellar neuropile. No ocellar processes appear to be given off to the corpora pedunculata, centra! body, nervi corporis cardiaci, antenna! lobes, or circumesophageal connectives; it is uncertain whether ocellar collaterals extend into the protocerebral bridge or optic lobes. Cell bodies of giant and medium-sized fibers are located in the pars intercerebralis.     

The developmental expression of serotonin-immunoreactivity in the brain of the pupal honeybee

The biogenic amine serotonin is a neurotransmitter and modulator in both vertebrates and invertebrates. In the CNS of insects, serotonin is expressed by identifiable subsets of neurons. In this paper, we characterize the onset of expression in the brain and suboesophageal ganglion of the honeybee during pupal development. Several identified serotonin-immunoreactive neurons are present in the three neuromeres of the suboesophageal ganglion the dorsal protocerebrum, and the deutocerebrum at pupal ecdysis. Further immunoreactive neurons are incorporated into the developing pupal brain in two characteristic developmental phases. During the first phase, 5 days after pupal ecdysis, serotonin immunoreactivity is formed in the protocerebral central body, the lamina and lobula, and the deutocerebral antennal lobe. During the second phase, 2 days later, immunoreactivity appears in neurons of the protocerebral noduli of the central complex, the medulla, and the pedunculi and lobes of the mushroom bodies. Three novel serotonin-immunoreactive neurons that innervate the central complex and the mushroom bodies can be individually identified.     

Morphology of the larval and adult brains of the monarch butterfly, Danaus plexippus plexippus, L

Cell population and neuropile morphology of larval and adult brains of the monarch butterfly, Danaus plexippus plexippus, L., are compared. The larval brain is in continuous transition, the processes of adult brain development being underway from the earliest larval stages. It is characterized by a less diverse population of cells and more homogenous fiber areas than those of the adult. Neuroblasts, which divide to form the neurones of the adult brain, occur either in discrete proliferation centers or scattered among the larval ganglion cells. The larval brain contains, in addition to small homogeneous antennal centers and a distinct larval optic center, rapidly developing adult optic centers, corpora pedunculata, and protocerebral bridge. The larval brain lacks a central body. Major differences between larval and adult brains are clearly related to the increased dependence of the adult upon sensory input from the eyes and antennae.     

Processing of antennal information in extrinsic mushroom body neurons of the bee brain

Summary In the bee brain neural activity of interneurons of the inner antenno-cerebral tract (inputs to the mushroom body) and extrinsic neurons of the-lobe (output cells) was recorded intracellularly. The cells were stained with Lucifer Yellow. The response characteristics of the neurons to light, various antennal stimuli and mechanical stimuli to thorax and abdomen were studied.The cells of the inner antenno-cerebral tract (ACT) have uniglomerular dendritic arborizations in the antennal lobe and send projections into the calyces of the ipsilateral mushroom body and the lateral protocerebral lobe. 93% of the neurons are bi- or multimodal. No responses to light stimuli were found. Tactile stimuli to the antennae are only effective when applied ipsilaterally. Only one neuron showed marked differences in the responses to the qualitative testing of three odors: rose, lavender and isoamyl acetate.The cells can be classified according to their response characteristics; the following response types were found: (1) inhibitory responses to the stimuli, (2) inhibitory responses to olfactory and excitatory responses to mechanical stimuli or vice versa, (3) excitatory responses to mechanical and sugar water stimuli, (4) excitation to olfactory stimuli and to touching the antenna with a drop of water or sugar water, (5) excitation to mechanical stimuli to head, thorax and abdomen and inhibition to sugar water stimuli.The recorded extrinsic-lobe neurons have small dendritic bands perpendicular to the Kenyon cells, their axons project to the contralateral median protocerebrum. These cells have ipsilateral antennal and mostly ipsilateral optic inputs and process information from thoracic and abdominal mechanoreceptors. All responses are excitatory.The recordings suggest that the mushroom bodies are multimodal integration centers, where antennal information is first combined with visual inputs.Abbreviation ACT antenno-cerebral tract     

5-HT-like immunoreactivity in the central nervous system of the crayfish,Pacifastacus leniusculus

An immunocytochemical technique with the use of three different antibodies raised against serotonin was applied to localize the immunoreactive neurons in the central nervous system of the crayfish, Pacifastacus leniusculus. Immunoreactive neurons were found in three optic ganglia (medulla externa, interna and terminalis). They appeared in three layers of the medulla externa and interna. The medulla terminalis displayed three prominent groups of immunoreactive perikarya and mainly marginal immunoreactive fibres. Immunoreactive areas of the brain comprised the protocerebral bridge, central body, paracentral lobes and two loci in the anterior portion of the protocerebrum, i.e., the terminal areas for immunoreactive fibres from the optic centres. The olfactory lobes showed a specific immunoreactive pattern. In addition, diffusely and sparsely distributed immunoreactive fibres were found throughout the brain. The immunoreactive neurons are largely localized in the same areas of the central nervous system as the catecholaminergic neurons although some distinct differences occur.     

Neurophysiology of the vibration sense in locusts and bushcrickets: The responses of ventral-cord neurones

In the locustid Locusta migratoria and the tettigoniids Decticus verrucivorus and Tettigonia cantans, comparative aspects of physiological properties of vibratory/auditory ventral-cord neurones were studied by single cell recordings.These neurones all receive inputs from both vibratory and auditory receptors. Nevertheless, they can be classified into “V neurones” responding preferentially to vibration stimuli, “VS neurones” responding to vibration and airborne sound, and “S neurones” responding preferentially to airborne sound. In every group, there are several types with different physiological properties, normally represented by one neurone on each body side.In Locusta and in the tettigoniid species, the same physiological types of vibratory/auditory neurones were found, although there are differences in the synaptic connectivity of the vibration receptors of the different legs. In Locusta, the middle leg receptors have the strongest influence on the generation of suprathreshold responses of the central neurones, whereas in the tettigoniids the receptors of the ipsilateral fore leg are the most influential.Two of the V neurones receive inputs mainly from campaniform sensilla and other low-frequency vibration receptors, the other V and VS neurones are mainly influenced by the subgenual receptors. Central information processing results in preferential responses to different frequency/intensity ranges in different neurones.Most VS neurone types show the same response characteristics (e.g. time pattern of response, habituation) either to vibration or to airborne-sound stimuli. Simultaneous presentation of both stimuli leads to qualitative changes in the response characteristics. Therefore, the co-processing of auditory and vibratory signals seems to be very important in the acoustic behaviour of grasshoppers.     

Serotonin immunoreactive interneurons in the brain of the Remipedia: new insights into the phylogenetic affinities of an enigmatic crustacean taxon

ABSTRACT: BACKGROUND: Remipedia, a group of homonomously segmented, cave-dwelling, eyeless arthropods have been regarded as basal crustaceans in most early morphological and taxonomic studies. However, molecular sequence information together with the discovery of a highly differentiated brain led to a reconsideration of their phylogenetic position. Various conflicting hypotheses have been proposed including the claim for a basal position of Remipedia up to a close relationship with Malacostraca or Hexapoda. To provide new morphological characters that may allow phylogenetic insights, we have analyzed the architecture of the remipede brain in more detail using immunocytochemistry (serotonin, acetylated alpha-tubulin, synapsin) combined with confocal laser-scanning microscopy and image reconstruction techniques. This approach allows for a comprehensive neuroanatomical comparison with other crustacean and hexapod taxa. RESULTS: The dominant structures of the brain are the deutocerebral olfactory neuropils, which are linked by the olfactory globular tracts to the protocerebral hemiellipsoid bodies. The olfactory globular tracts form a characteristic chiasm in the center of the brain. In Speleonectes tulumensis, each brain hemisphere contains about 120 serotonin immunoreactive neurons, which are distributed in distinct cell groups supplying fine, profusely branching neurites to 16 neuropilar domains. The olfactory neuropil comprises more than 300 spherical olfactory glomeruli arranged in sublobes. Eight serotonin immunoreactive neurons homogeneously innervate the olfactory glomeruli. In the protocerebrum, serotonin immunoreactivity revealed several structures, which, based on their position and connectivity resemble a central complex comprising a central body, a protocerebral bridge, W-, X-, Y-, Z-tracts, and lateral accessory lobes. CONCLUSIONS: The brain of Remipedia shows several plesiomorphic features shared with other Mandibulata, such as deutocerebral olfactory neuropils with a glomerular organization, innervations by serotonin immunoreactive interneurons, and connections to protocerebral neuropils. Also, we provided tentative evidence for W-, X-, Y-, Z-tracts in the remipedian central complex like in the brain of Malacostraca, and Hexapoda. Furthermore, Remipedia display several synapomorphies with Malacostraca supporting a sister group relationship between both taxa. These homologies include a chiasm of the olfactory globular tract, which connects the olfactory neuropils with the lateral protocerebrum and the presence of hemiellipsoid bodies. Even though a growing number of molecular investigations unites Remipedia and Cephalocarida, our neuroanatomical comparison does not provide support for such a sister group relationship.     

Convergence of heterotopic nociceptive information onto neurons of the subnucleus reticularis dorsalis in the rat

The nature of stimuli that activate neurones located within the Subnucleus Reticularis Dorsalis (SRD) was determined. These neurones do not respond to visual, auditory or proprioceptive stimuli but are preferentially or even exclusively activated by noxious stimuli applied to any part of the body; only neurones of a sub-group have a predominantly contralateral activation. All the neurones were exclusively activated by stimulation of A delta or A delta and C afferent fibres. These observations suggest that this well-delimited reticular structure is specifically involved in the processing of nociceptive information.