Rudolf Loesel

Pygmy squids and giant brains: mapping the complex cephalopod CNS by phalloidin staining of vibratome sections and whole-mount preparations.

Among bilaterian invertebrates, cephalopod molluscs (e.g., squids, cuttlefish and octopuses) have a central nervous system (CNS) that rivals in complexity that of the phylogenetically distant vertebrates (e.g., mouse and human). However, this prime example of convergent evolution has rarely been the subject of recent developmental and evolutionary studies, which may partly be due to the lack of suitable neural markers and the large size of cephalopod brains. Here, we demonstrate the usefulness of fluorescence-coupled phalloidin to characterize the CNS of cephalopods using histochemistry combined with confocal laser scanning microscopy. Whole-mount preparations of developmental stages as well as vibratome sections of embryonic and adult brains were analyzed and the benefits of this technique are illustrated. Compared to classical neuroanatomical and antibody-based studies, phalloidin labeling experiments are less time-consuming and allow a high throughput of samples. Besides other advantages summarized here, phalloidin reliably labels the entire neuropil of the CNS of all squids, cuttlefish and octopuses investigated. This facilitates high-resolution in toto reconstructions of the CNS and contributes to a better understanding of the organization of neural networks. Amenable for multi-labeling experiments employing antibodies against neurotransmitters, proteins and enzymes, phalloidin constitutes an excellent neuropil marker for the complex cephalopod CNS.

Immunofluorescence analysis of the internal brain anatomy of Nereis diversicolor (Polychaeta, Annelida)

Comparative analyses of neuroanatomical characters can make valuable contributions to the inference of phylogenetic relationships. Whereas investigations in this field are numerous for arthropods, in-depth studies on other protostomes are sparse. Here, we provide a survey of the internal neuroarchitecture of the brain of the aciculate ragworm Nereis diversicolor (Polychaeta, Annelida). Descriptions are based on confocal laser scanning microscope analyses of brain sections labeled with the nuclear marker DAPI and antibodies raised against FMRF-amide, serotonin, and histamine. Autofluorescence of the nervous tissue has been utilized to further elucidate the anatomical structures of the brain. The architecture of two major brain compartments, i.e., the paired mushroom bodies and the central optic neuropil, is described in detail. The findings are compared with existent literature on polychaete neuroanatomy and on arthropod neuroanatomy, and possible phylogenetic implications are outlined.

Three-dimensional reconstruction of mushroom body neuropils in the polychaete species Nereis diversicolor and Harmothoe areolata (Phyllodocida, Annelida)

Mushroom bodies are prominent brain neuropils present in most arthropod representatives. Similar structures in the brain of certain polychaete species are possibly homologous to these structures. Using three-dimensional reconstruction techniques, we investigated the structural composition of the mushroom body neuropils in the polychaete species Nereis diversicolor and Harmothoe areolata. Comparative analysis revealed a common organization of neuropil substructures in both species that closely matches the basic assembly of arthropod mushroom bodies. Concurring with earlier homology assessments, these neuroarchitectural similarities provide support for a common origin of mushroom body neuropils in polychaetes and arthropods. Beyond that, differences in the morphological differentiation of neuropil substructures indicate polychaete mushroom bodies to show a high degree of morphological variability, thus impeding the quest for a common ground pattern of these brain centers.

A simple fluorescent double staining method for distinguishing neuronal from non-neuronal cells in the insect central nervous system.

Being able to discriminate between neurons and non-neuronal cells such as glia and tracheal cells has been a major problem in insect neuroscience, because glia-specific antisera are available for only a small number of species such as Drosophila melanogaster and Manduca sexta. Especially developmental or comparative studies often require an estimate of neuron numbers. Since neuronal and glial cell bodies are in many cases indiscernible in situ, a method to distinguish neurons from non-neuronal cells that works in any given species is wanting. Another application is cell culturing. Cultured cells usually change their outward shape dramatically after being isolated so that it is frequently impossible to tell neurons and glia apart. Here, we present a simple method that uses a commercially available antiserum directed against horseradish peroxidase, which specifically stains neurons but no other cell type in every insect species investigated. Counterstaining with DAPI, a fluorescent chromophore that binds to double-stranded DNA in the nuclei of all cells, yields the total number of cells in a given sample. Thus, double labeled cells can be identified as neurons, cells that carry only DAPI staining are non-neuronal.

Architectural Principles and Evolution of the Arthropod Central Nervous System

This is an exciting time for arthropod neuroanatomists! A wealth of reviews, special issues, book chapters, and entire book volumes published during the last 10 years shows the unbroken interest in and enthusiasm for the arthropod nervous system and for gaining insights into its architecture, physiology, and aspects of neuroethology (Barth and Schmid 2001; Wiese 2001, 2002; Barth 2002; North and Greenspan 2007; Breithaupt and Thiel 2011, Galizia et al. 2012; Land and Nilsson 2012; Strausfeld 2012). Numerous review articles and book chapters witness that neurobiology is one of the most active fields of arthropod research. Recently, featured topics are, for example, the crustacean central nervous system (Schmidt and Mellon 2011; Harzsch et al. 2012; Sandeman et al. in press), structure and function of crustacean chemosensory sensilla (e.g. Hallberg and Skog 2011; Mellon and Reidenbach 2011), chelicerate strain detection systems (Barth 2012), and insect olfaction (Galizia and Szyska 2008; Hansson and Stensmyr 2011; Hansson et al. 2011; Sachse and Krieger 2011). Moreover, the central nervous system and visual organs of neglected taxa such as Myriapoda (Sombke et al. 2011a, 2012), Onychophora (Mayer 2006; Strausfeld et al. 2006a, b; Eriksson and Stollewerk 2010; Whitington and Mayer 2011), Trilobita (Clarkson et al. 2006), and Xiphosura (Battelle 2006) have been analyzed with contemporary techniques.

Lophotrochozoan neuroanatomy: An analysis of the brain and nervous system of Lineus viridis(Nemertea) using different staining techniques.

BackgroundThe now thriving field of neurophylogeny that links the morphology of the nervous system to early evolutionary events relies heavily on detailed descriptions of the neuronal architecture of taxa under scrutiny. While recent accounts on the nervous system of a number of animal clades such as arthropods, annelids, and molluscs are abundant, in depth studies of the neuroanatomy of nemerteans are still wanting. In this study, we used different staining techniques and confocal laser scanning microscopy to reveal the architecture of the nervous system of Lineus viridis with high anatomical resolution.ResultsIn L. viridis, the peripheral nervous system comprises four distinct but interconnected nerve plexus. The central nervous system consists of a pair of medullary cords and a brain. The brain surrounds the proboscis and is subdivided into four voluminous lobes and a ring of commissural tracts. The brain is well developed and contains thousands of neurons. It does not reveal compartmentalized neuropils found in other animal groups with elaborate cerebral ganglia.ConclusionsThe detailed analysis of the nemertean nervous system presented in this study does not support any hypothesis on the phylogenetic position of Nemertea within Lophotrochozoa. Neuroanatomical characters that are described here are either common in other lophotrochozoan taxa or are seemingly restricted to nemerteans. Since detailed descriptions of the nervous system of adults in other nemertean species have not been available so far, this study may serve as a basis for future studies that might add data to the unsettled question of the nemertean ground pattern and the position of this taxon within the phylogenetic tree.

Neurophylogeny: Retracing Early Metazoan Brain Evolution

The current view of early metazoan phylogeny suggests that the bilaterian body plan arose only once during evolution. This first urbilaterian animal was most likely equipped with an anterior condensation of nerve cells – a brain – from which all brains of modern animals have diverged. Until recently, the ancestor of all bilaterian phyla was viewed as a very simple animal with an accordingly simple brain. Molecular studies, however, demonstrate a multitude of homologous genes that are expressed in similar patterns in the developing brains of vertebrates, insects, and annelids. Taken together, these findings imply that the anatomy of the urbilaterian cerebrum might have been more elaborate than previously assumed. If true, ancient architectural features might have been conserved during evolution and should be identifiable in distantly related modern animal phyla. Comparative studies on representatives of arthropods, onychophorans, and annelids suggest that this is indeed the case. This chapter summarizes recent neuroanatomical surveys that aim to retrace the early evolution of the metazoan brain and to use neuroanatomical data to test conflicting hypothesis on phylogenetic relationships between major animal phyla.