Protocerebral Tract Research Articles

Abstract concept learning in a simple neural network inspired by the insect brain.

The capacity to learn abstract concepts such as ‘sameness’ and ‘difference’ is considered a higher-order cognitive function, typically thought to be dependent on top-down neocortical processing. It is therefore surprising that honey bees apparantly have this capacity. Here we report a model of the structures of the honey bee brain that can learn sameness and difference, as well as a range of complex and simple associative learning tasks. Our model is constrained by the known connections and properties of the mushroom body, including the protocerebral tract, and provides a good fit to the learning rates and performances of real bees in all tasks, including learning sameness and difference. The model proposes a novel mechanism for learning the abstract concepts of ‘sameness’ and ‘difference’ that is compatible with the insect brain, and is not dependent on top-down or executive control processing.

Three-dimensional organization of the brain and distribution of serotonin in the brain and ovary, and its effects on ovarian steroidogenesis in the giant freshwater prawn, Macrobrachium rosenbergii.

The giant freshwater prawn, Macrobrachium rosenbergii, is an economically important crustacean species which has also been extensively used as a model in neuroscience research. The crustacean central nervous system is a highly complex structure, especially the brain. However, little information is available on the brain structure, especially the three-dimensional organization. In this study, we demonstrated the three-dimensional structure and histology of the brain of M. rosenbergii together with the distribution of serotonin (5-HT) in the brain and ovary as well as its effects on ovarian steroidogenesis. The brain of M. rosenbergii consists of three parts: protocerebrum, deutocerebrum and tritocerebrum. Histologically, protocerebrum comprises of neuronal clusters 6-8 and prominent anterior and posterior medial protocerebral neuropils (AMPN/PMPN). The protocerebrum is connected posteriorly to the deutocerebrum which consists of neuronal clusters 9-13, medial antenna I neuropil, a paired lateral antenna I neuropils and olfactory neuropils (ON). Tritocerebrum comprises of neuronal clusters 14-17 with prominent pairs of antenna II (AnN), tegumentary and columnar neuropils (CN). All neuronal clusters are paired structures except numbers 7, 13 and 17 which are single clusters located at the median zone. These neuronal clusters and neuropils are clearly shown in three-dimensional structure of the brain. 5-HT immunoreactivity (-ir) was mostly detected in the medium-sized neurons and neuronal fibers of clusters 6/7, 8, 9, 10 and 14/15 and in many neuropils of the brain including anterior/posterior medial protocerebral neuropils (AMPN/PMPN), protocerebral tract, protocerebral bridge, central body, olfactory neuropil (ON), antennal II neuropil (Ann) and columnar neuropil (CN). In the ovary, the 5-HT-ir was light in the oocyte step 1(Oc1) and very intense in Oc2-Oc4. Using an in vitro assay of an explant of mature ovary, it was shown that 5-HT was able to enhance ovarian estradiol-17β (E2) and progesterone (P4) secretions. We suggest that 5-HT is specifically localized in specific brain areas and ovary of this prawn and it plays a pivotal role in ovarian maturation via the induction of female sex steroid secretions, in turn these steroids may enhance vitellogenesis resulting in oocyte growth and maturation.

Distribution of serotonin and dopamine in the central nervous system of the female mud crab, Scylla olivacea (Herbst)

In crustaceans serotonin (5-HT) and dopamine (DA) are neurotransmitters that play roles in the modulation of numerous physiological functions, including reproduction. However, in the mud crab, Scylla olivacea, the distributions of 5-HT and DA in the CNS have not yet been investigated. The aim of our study was to map the distributions of these two neurotransmitters in the central nervous system (CNS) of the female of this crab during the late stage of ovarian development. We found 5-HT immunoreactivity (-ir) and DA-ir in many parts of the CNS, including the eyestalk, brain, and thoracic ganglia. In the eyestalk, 5-HT-ir was localized in the medulla terminalis (MT), hemi-ellipsoid body (HB), and protocerebral tract (PT), whereas DA-ir was present in neuronal cluster 1, the LG neuropils, and PT. In the brain, 5-HT-ir and DA-ir were detected in cells and fibers of neuronal clusters 6, 7, 8, 9, 10, 11, 14, and 15. In the ventral nerve cord, 5-HT-ir was present in neurons of the abdominal ganglia, whereas DA was only present in fibers. These spatial distributions of 5-HT and DA suggest that they may be involved in the neuromodulation of important physiological functions, including ovarian maturation, as shown in other non-crab decapods.

The crustacean central nervous system in focus: subacute neurodegeneration induces a specific innate immune response.

To date nothing is known about the subacute phase of neurodegeneration following injury in invertebrates. Among few clues available are the results published by our group reporting hemocytes and activated glial cells at chronic and acute phases of the lesion. In vertebrates, glial activation and recruitment of immunological cells are crucial events during neurodegeneration. Here, we aimed to study the subacute stage of neurodegeneration in the crab Ucides cordatus, investigating the cellular/molecular strategy employed 48 hours following ablation of the protocerebral tract (PCT). We also explored the expression of nitric oxide (NO) and histamine in the PCT during this phase of neurodegeneration. Three immune cellular features which seem to characterize the subacute phase of neurodegeneration were revealed by: 1) the recruitment of granulocytes and secondarily of hyalinocytes to the lesion site (inducible NO synthase- and histamine-positive cells); 2) the attraction of a larger number of cells than observed in the acute phase; 3) the presence of activated glial cells as shown by the round shaped nuclei and increased expression of glial fibrillary acidic protein. We suggest that molecules released from granulocytes in the acute phase attract the hyalinocytes thus moving the degeneration process to the subacute phase. The importance of our study resides in the characterization of cellular and biochemical strategies peculiar to the subacute stage of the neurodegeneration in invertebrates. Such events are worth studying in crustaceans because in invertebrates this issue may be addressed with less interference from complex strategies resulting from the acquired immune system.

Dual olfactory pathway in Hymenoptera: Evolutionary insights from comparative studies

In the honeybee ( Apis mellifera) and carpenter ant ( Camponotus floridanus) the antennal lobe output is connected to higher brain centers by a dual olfactory pathway. Two major sets of uniglomerular projection neurons innervate glomeruli from two antennal-lobe hemispheres and project via a medial and a lateral antennal-lobe protocerebral tract in opposite sequence to the mushroom bodies and lateral horn. Comparison across insects suggests that the lateral projection neuron tract represents a special feature of Hymenoptera. We hypothesize that this promotes advanced olfactory processing associated with chemical communication, orientation and social interactions. To test whether a dual olfactory pathway is restricted to social Hymenoptera, we labeled the antennal lobe output tracts in selected species using fluorescent tracing and confocal imaging. Our results show that a dual pathway from the antennal lobe to the mushroom bodies is present in social bees, basal and advanced ants, solitary wasps, and in one of two investigated species of sawflies. This indicates that a dual olfactory pathway is not restricted to social species and may have evolved in basal Hymenoptera. We suggest that associated advances in olfactory processing represent a preadaptation for life styles with high demands on olfactory discrimination like parasitoism, central place foraging, and sociality.

Identity of the cells recruited to a lesion in the central nervous system of a decapod crustacean

In a previous study, we analyzed and described the features of the degeneration of the protocerebral tract (PCT) of the crustacean Ucides cordatus, after the extirpation of the eyestalk. In that study, among axons with axoplasmic degeneration, cells with granules resembling blood cells (hemocytes) were seen. Therefore, in the present study, we characterized the circulating hemocytes and compared them with the cells recruited to a lesion, which was produced as in the former study. Using histochemistry, immunohistochemistry, and electron microscopy (transmission and scanning), we confirmed that circulating and recruited cells display a similar morphology. Therefore, in the crab, hemocytes were attracted to the lesion site in the acute stage of degeneration, appearing near local glial cells that showed signs of being responsive. Some of the attracted hemocytes displayed a morphology that was considered to be possibly activated blood cells. Also, the cells that migrated to the injured PCT displayed features, such as the presence of hydrolytic enzymes and an ability to phagocytize neural debris, similar to those of vertebrates. In summary, our results indicate that hemocytes were not only phagocytizing neural debris together with glial cells but also that they may be concerned with creating a favorable environment for regenerating events.

Sensory Activation and Receptive Field Organization of the Lateral Giant Escape Neurons in Crayfish

Crayfish (Procambarus clarkii) have bilateral pairs of giant interneurons that control rapid escape movements in response to predatory threats. The medial giant neurons (MGs) can be made to fire an action potential by visual or tactile stimuli directed to the front of the animal and this leads to an escape tail-flip that thrusts the animal directly backward. The lateral giant neurons (LGs) can be made to fire an action potential by strong tactile stimuli directed to the rear of the animal, and this produces flexions of the abdomen that propel the crayfish upward and forward. These observations have led to the notion that the receptive fields of the giant neurons are locally restricted and do not overlap with each other. Using extra- and intracellular electrophysiology in whole animal preparations of juvenile crayfish, we found that the receptive fields of the LGs are far more extensive than previously assumed. The LGs receive excitatory inputs from descending interneurons originating in the brain; these interneurons can be activated by stimulation of the antenna II nerve or the protocerebral tract. In our experiments, descending inputs alone could not cause action potentials in the LGs, but when paired with excitatory postsynaptic potentials elicited by stimulation of tail afferents, the inputs summed to yield firing. Thus the LG escape neurons integrate sensory information received through both rostral and caudal receptive fields, and excitatory inputs that are activated rostrally can bring the LGs' membrane potential closer to threshold. This enhances the animal's sensitivity to an approaching predator, a finding that may generalize to other species with similarly organized escape systems.

Sensory Representation and Learning-Related Plasticity in Mushroom Body Extrinsic Feedback Neurons of the Protocerebral Tract

Gamma-aminobutyric acid immunoreactive feedback neurons of the protocerebral tract are a major component of the honeybee mushroom body. They have been shown to be subject to learning-related plasticity and provide putative inhibitory input to Kenyon cells and the pedunculus extrinsic neuron, PE1. We hypothesize, that learning-related modulation in these neurons is mediated by varying the amount of inhibition provided by feedback neurons. We performed Ca2+ imaging recordings of populations of neurons of the protocerebral-calycal tract (PCT) while the bees were conditioned in an appetitive olfactory paradigm and their behavioral responses were quantified using electromyographic recordings from M17, the muscle which controls the proboscis extension response. The results corroborate findings from electrophysiological studies showing that PCT neurons respond to sucrose and odor stimuli. The odor responses are concentration dependent. Odor and sucrose responses are modulated by repeated stimulus presentations. Furthermore, animals that learned to associate an odor with sucrose reward responded to the repeated presentations of the rewarded odor with less depression than they did to an unrewarded and a control odor.

Electron microscopy and morphometric analyses of microtubules in two differently sized types of axons in the protocerebral tract of a crustacean

Despite several reports on the morphology and functions associated with the morphometry of the vertebrate axoplasm cytoskeleton, the subject has not been thoroughly explored in invertebrates. In vertebrates, among many other functions, microtubules (MTs) serve as scaffolding for axon assembly, and neurofilaments (NFs) as the elements that determine the axon caliber. Intermediate filaments have never been described by electron microscopy in arthropods, although NF proteins have been revealed in the MT side-arms of the axoplasm of certain species, such as the crab Ucides cordatus. Thus, it is not known which elements of the cytoskeleton of invertebrates are responsible for determination of the axon caliber. We studied, by electron microscopy and morphometric analyses, the MT and axon area variability in differently sized axons of the protocerebral tract of the crab Ucides cordatus. Our results revealed differences in the distance between MTs, in MT density and number, and in the areas of differently sized axons. The number of MTs increases with the axon area, but this relationship is not directly proportional. Therefore, MT density is greater in smaller axons than in medium axons, similar to the morphometry of the vertebrate axon MT. The distance between MTs is, however, directly related to the axonal area. On the basis of the results shown here, and on previous reports by us and others, we suggest that MTs may be involved in the determination of the axon caliber, possibly due to the presence of NF proteins found in the side-arms.

Ultrastructural Study of Normal and Degenerating Nerve Fibers in the Protocerebral Tract of the Crab Ucides cordatus

Wallerian degeneration is a very well described phenomenon in the vertebrate nervous system. In arthropods, and especially in crustaceans, nerve fiber degeneration has not been described extensively. In addition, literature shows that the events do not follow the same patterns as in vertebrates. In this study we report, by qualitative and quantitative ultrastructural analyses, the features and time course of the protocerebral tract degeneration following extirpation of the optic stalk. No remarkable changes were observed seven days after lesion. After 28 days the protocerebral tracts presented apparently preserved small and large diameter axons and some degenerating medium axons, with irregular contours and empty-looking aspect of the axoplasm. Forty days after the ablation of the optic stalks, both small (type I) and medium (type II and III) axons revealed signs of partial or total degeneration, but large nerve fibers (type IV) were still intact. After 45 days, the tract showed signs of advanced stage of degeneration and, apart from large axons, normal-looking fibers were almost absent. At these 3 last time points, degenerating axons displayed different electron densities and aspects, probably correlating to different onset times of the process. In addition, cells with granules in their cytoplasm, possibly hemocytes, were quite distinct, especially at 40 and 45 days after axotomy. These cells might share with glial cells the function of phagocytosis of cellular debris during the protocerebral tract degeneration. Quantitative analysis showed that the number of degenerating fibers increased significantly from 28 to 40 days after lesion, whereas the number of normal fibers decreased accordingly. Measurements of cross-sectional areas of normal and degenerating axons showed that types II and III (medium) start to degenerate before type I (small). Type IV (large) axons do not degenerate, even after 40 days. Therefore, we can conclude that degeneration in these afferent fibers starts late after axotomy, but proceeds at a faster rate afterwards until the complete degeneration of small and medium axons.

Identification of a neurofilament-like protein in the protocerebral tract of the crab Ucides cordatus.

Neurofilaments (NFs) have not been observed in crustaceans using conventional electron microscopy, and intermediate filaments have never been described in crustaceans and other arthropods by immunocytochemistry. Since polypeptides, labeled by the NN18-clone antibody, were revealed on microtubule side-arms of crayfish, we have tested, in this study, whether proteins similar to mammalian NFs are present in the protocerebral tract (PCT) of the crab Ucides cordatus. We used immunohistochemistry for light microscopy with monoclonal antibodies against three different NF subunits, high (NF-H), medium (NF-M), and light (NF-L). Labeling was observed with the NN18-clone, which recognizes NF-M. In order to confirm the results obtained with the immunohistochemical reactions, Western blotting, using the three primary antibodies, was performed and the presence of NF-M was confirmed. The NN18-clone monoclonal antibody recognized a protein of approximately 160 kDa, similar to the mammalian NF-M protein, but NF-L and NF-H were not recognized. Conventional transmission electron microscopy was used to observe the ultrastructural components of the axons and immunoelectron microscopy was used to show the distribution of the NF-M-like polypeptides along cytoskeletal elements of the PCT. Our results agree with previous studies on crustacean NF proteins that have reported negative immunoreactions against NF-H and NF-L subunits and positive immunoreactions against the mammalian NF-M subunit. However, the protein previously referred to as P600 and recognized by the NN18-clone, has a very high molecular weight, thus, being different from mammalian NF-M subunit and from the protein revealed now in our study.

Physiology and morphology of visual movement detector neurons in a crab (Decapoda: Brachyura).

Although visually elicited behaviors have been extensively studied in crabs, their investigation at the neurophysiological level is scant. The present study is a physiological and morphological description of intracellularly recorded and dye injected visual movement detector neurons that respond to the same stimulus that elicits the escape response in the crab Chasmagnathus granulatus. The neurons were investigated in intact animals. The response of movement detector neurons to the danger stimulus (an object moving above the animal) consists of a strong discharge of action potentials frequently superimposed on noisy graded potentials, whereas the response to stationary changes in illumination is weak or undetectable. The response to the moving stimulus is relatively independent of the background intensity and of the contrast between target and background. Repeated presentations of the moving stimulus produce rapid habituation of the neural response. Some of the neurons also respond to mechanical stimulation. These physiological results coincide with those from early studies on visual movement detector fibers of crustaceans achieved by extracellular recordings. However, there are no previous morphological studies of these neurons. Intracellular injection with Lucifer Yellow revealed that these neurons in Chasmagnathus arborize extensively in the internal medulla and in the lateral protocerebrum. They have their somata located in the cell body cluster laying beneath the internal medulla. Their axons project centripetally across the protocerebral tract.

Electron microscopy of glial cells of the central nervous system in the crab Ucides cordatus.

Invertebrate glial cells show a variety of morphologies depending on species and location. They have been classified according to relatively general morphological or functional criteria and also to their location. The present study was carried out to characterize the organization of glial cells and their processes in the zona fasciculata and in the protocerebral tract of the crab Ucides cordatus. We performed routine and cytochemical procedures for electron microscopy analysis. Semithin sections were observed at the light microscope. The Thiéry procedure indicated the presence of carbohydrates, particularly glycogen, in tissue and in cells. To better visualize the axonal ensheathment at the ultrastructural level, we employed a method to enhance the unsaturated fatty acids present in membranes. Our results showed that there are at least two types of glial cells in these nervous structures, a light one and a dark one. Most of the dark cell processes have been mentioned in the literature as extracellular matrix, but since they presented an enveloping membrane, glycogen and mitochondria--intact and with different degrees of disruption--they were considered to be glial cells in the present study. We assume that they correspond to the perincurial cells on the basis of their location. The light cells must correspond to the periaxonal cells. Some characteristics of the axons such as their organization, ensheathment and subcellular structures are also described.

Glial Cells of the Central Nervous System in the Crab Ucides cordatus

Glial cells and their processes were characterized in the fasciculated zone and in the protocerebral tract of the crab Ucides cordatus by light and electron microscopy. Thiery and PAS procedures indicate the presence of carbohydrates, particularly glycogen in cells. Immunohistochemistry was used to observe tubulin distribution in the glial cells. Our results demonstrate at least two types of glial cells in the fasciculated zone and in the protocerebral tract, separable by their location and electron density. Judging by their position, electron-lucent cells may correspond to periaxonal cells and electron-dense ones may correspond to perineurial cells. The electron-dense processes have previously been interpreted as extracellular matrix, but since they feature an enveloping membrane and contain glycogen and mitochondria (intact and with varying degrees of disruption) we consider them to be part of one type of glial cells. Additional key words: Crustacea, Malacostraca, Brachyura, glia Invertebrate glial cells have traditionally been classified according to certain relatively general morphological or functional criteria and also by their anatomical position (Haimori & Horridge 1966; Radojcic & Pentreath 1979). Because invertebrates do not have true compact myelin, no glial class equivalent to vertebrate oligodendrocytes can be identified. In addition, immunocytochemical markers for vertebrate astrocytes in most cases fail to label invertebrate glia. (One of the exceptions is glutamine synthetase, a specific marker for astrocytes in vertebrates, which has also successfully labeled non-neuronal cells in lobster olfactory regions: Linser et al. 1997.) The glial cell types of arthropods share many morphological characteristics. First, in many species, the chromatin of glial cell nuclei is clumped in the periphery. This feature is rarely seen in neuronal nuclei. Second, mitochondria, endoplasmic reticulum, and Golgi structures are generally common. Third, between neighboring glial cells and also between glial cells and neurons, diverse membrane specializations are found (see Pentreath 1987). In the nervous system of some species of higher crustaceans, i.e., Decapoda, there is good anatomical and physiological evidence for an insulating sheath with nodes and rapid saltatory conduction (Heuser & Doggenweiler 1966). Where present, the sheath coma Author for correspondence. E-mail: sallodi@chagas.biof.ufrj.br prises numerous laminae, each containing cytoplasm and forming a seam. The nodes are loosely wrapped by a characteristic type of glial cell called the nodal cell. The axons can be wrapped either simply by a single glial process or in groups, by complex, tightly packed multiple layers, resembling vertebrate myelin (Bullock et al. 1977). Important differences from vertebrate myelin are: (a) the crustacean laminae do not connect to form a spiral; (b) the nuclei of the sheath cells lie on the inside of the sheath; (c) desmosomes join adjacent laminae; (d) sometimes an adjacent extracellular matrix may occur between neighboring glial cell processes (Heuser & Doggenweiler 1966). The participation of neuroglia in the blood-brain barrier is an important function of glial cells in both vertebrates and invertebrates (Abbott et al. 1986; Abbott 1995). Unlike the situation in higher vertebrates, in which the blood-brain barrier is formed by tight junctions of endothelial cells lining capillaries of the central nervous system (see Peters et al. 1991), the barrier in crustaceans is formed by glial perineurium surrounding the ganglia. The sealing function of the barrier is thought to be due to the junctions between the perineurial cells and the extracellular matrix, which may be expanded and contain collagen-like fibrils (Abbott 1972; see Pentreath 1987). The most widely accepted function of glial cells is that they interact metabolically with neurons, providing nutrients, exchanging metabolites, and removing catabolites. Such a role is supported by glial stores of glycogen This content downloaded from 157.55.39.231 on Wed, 05 Oct 2016 04:16:00 UTC All use subject to http://about.jstor.org/terms Allodi, Silva, & Taffarel and large numbers of vesicular and granular inclusions in the cytoplasm (Pentreath 1987; Tsacopoulos & Magistretti 1996). Our goal was to characterize the organization of glial cells and their processes by light microscopy (LM) and transmission electron microscopy (TEM) and to correlate those findings with the distribution of extracellular matrix in the fasciculated zone (FZ) and in the protocerebral tract (PT) of the crab Ucides cordatus. The FZ comprises retinular cell axons that run from the retina to the lamina ganglionaris (Bell & Lightner 1988) and the PT is the tract that links the terminal medulla and the hemiellipsoid body to the anterior medial protocerebral neuropil and other areas of the brain (Sandeman et al. 1992). These neuropils were chosen because they are primarily composed of axons and glial cells.

Immunocytochemical detection of acetylated alpha-tubulin and Drosophila synapsin in the embryonic crustacean nervous system.

The caridean shrimp Palaemonetes argentinus Nobili is well suited for studying developmental aspects of the crustacean nervous system due to its rapid embryonic development and short reproductive cycle. In the present paper, we demonstrate the pattern of central axonal pathways in embryos of this species by immunohistochemical detection of acetylated alpha-tubulin. Development of the neuropil was elucidated by using an antibody to a Drosophila synapsin. In the ventral nerve cord, the segmental axonal scaffold consists of the paired lateral connectives, a median connective, and the anterior and posterior commissures. Three nerve roots were found to branch off each ganglion anlage, i.e. the main segmental nerve root, a smaller posterior nerve and the intersegmental nerve. However, this pattern is different in the mandibular segment where no intersegmental nerve and only one commissure was encountered. The anterior part of the brain consists of a tritocerebral and a deutocerebral anlage as well asthe anlage of the medial protocerebrum. The latter is connected to the eyestalk via the protocerebral tract. The sequence of development of the eyestalk ganglia was demonstrated in specimens which were stained with the anti-synapsin antibody. The medulla terminalis and medulla interna are the first neuropils to appear and are still fused in early stages. Later, the medulla interna splits off the medulla terminalis. The lamina ganglionaris is the last of the eyestalk neuropils to develop. These findings prove that immunocytochemistry against acetylated alpha-tubulin and synapsin are valuable tools for studying the development of the crustacean nervous system.

Characterization of Oscillatory Olfactory Interneurones in the Protocerebrum of the Crayfish

ABSTRACT We obtained intracellular electrophysiological recordings from local interneurones within the hemi-ellipsoid neuropile of the brain in the freshwater crayfish Cherax destructor and Procambarus clarkii. The recordings were made from perfused, isolated head preparations that provided several indications of a healthy physiological condition. The hemi-ellipsoid interneurones are spontaneously active, generating bursts of action potentials at regular intervals. The inter-burst period differs among neurones, varying from about 1.0 s at the shortest periods to around 30 s for the longest periods. Evidence from both electrophysiological recordings and from injection of Lucifer Yellow and Neurobiotin dyes into hemi-ellipsoid interneurones suggests that some of the cells in the populations are electrically coupled to one another. Hemi-ellipsoid interneurones are driven postsynaptically by axons within the lateral protocerebral tract. Experiments with focal electrical stimulation strongly suggest that the pathways responsible include axons of the olfactory–globular tract. These findings support our previous electron microscopical data showing that olfactory–globular tract axons are presynaptic to the hemi-ellipsoid interneurones. These findings support the conclusion that hemi-ellipsoid interneurones are an integral link in the central olfactory pathway of the crayfish.