Single Nerve Cells Research Articles

Morphological features of the pons in human fetuses 14-15 weeks of intrauterine development

Knowledge of human embryonic development is essential to improve our understanding of human fetal anatomy and for better understanding the etiology of congenital malformations. Currently, the structures of the posterior cranial fossa and the brain stem are of great interest to researchers, because of a large number of nuclei are located in the pons area, which play an important role in ensuring vital functions. The aim of the scientific work is to establish the morphological features of the pons of human fetuses at 14-15 weeks of gestation, the size and area of the nuclei of cranial nerves and neurons which are located in the area of the pons. Anatomical and histological research was carried out on 6 human fetuses aged 14-15 weeks of gestation. The cadaver material for research was obtained as a result of late abortions which were conducted according to medical indications in Vinnytsia maternity hospitals. Preparations were fixed in a 10 % solution of neutral formalin, stained with hematoxylin-eosin, toluidine blue modified by Nissl. Computer histometry (Toup View) was used for morphometric research. Statistical digital data were processed on a personal computer using Microsoft Excel 2016 and “Statistica 6.1” software. We established that in human fetuses at 14-15 weeks of intrauterine development, the nuclei of the trigeminal, abductor and vestibulocochlear nerves were detected. The nucleus of the facial nerve on 14-15 weeks of gestation is represented by single polygonal nerve cells with eosinophilic cytoplasm. The nucleus of the abductor nerve had the largest area, the cochlear nucleus of the vestibulocochlear nerve had the smallest area. The largest area of neurons was in the nuclei of the VIII pair of cranial nerve, the smallest – in the nucleus of the abductor and facial nerves. At 14-15 weeks of intrauterine development of human fetuses, the area of the cell nucleus could be determined only in the nuclei of the vestibulocochlear nerve, while the cell nuclei of the trigeminal, facial, and abductor nerves were represented by nerve cells of a spherical shape with a nucleus in which a basophilic nucleolus and a homogeneous eosinophilic cytoplasm were noted. Thus, in human fetuses of 14-15 weeks of intrauterine development, differences in the sizes and areas of the nuclei of cranial nerves and neurons that form nuclei in the pons area were found.

Preparation of Healthy Single Neuron or Astrocyte Suspension from Adult Mouse Brain for RNA-seq.

Single-cell RNA sequencing (scRNA-seq) has been widely applied in neuroscience research, enabling the investigation of cellular heterogeneity at the transcriptional level, the characterization of rare cell types, and the detailed analysis of the stochastic nature of gene expression. Isolation of single nerve cells in good health, especially from the adult rodent brain, is the most difficult and critical process for scRNA-seq. Here, we describe methods to optimize protease digestion of brain slices, which enable yield of millions of cells in good health from the adult brain.

Morphology and immunohistochemical characteristics of the otic ganglion in the chinchilla (Chinchilla laniger Molina).

The available literature provides relatively little information on the morphology of the autonomic head ganglia in rodents including their neurochemical codding. Morphological investigations of the otic ganglion of the chinchilla were performed using the modified acetylcholinesterase method. The cellular structure was investigated with histological techniques and neurochemical properties were studied with the double-labelling immunofluorescence method. Macromorphological investigations allowed the otic ganglion to be identified as a compact, oval agglomeration of neurons and nerve fibers. Multidimensional cross-sections revealed densely arranged neuronal perikarya and two populations of nerve cells differing in size were distinguished. The large cells (40-50 μm) accounted for about 80% of the neurons in the cross-sections. Moreover, a small number of intraganglionic nerve fibers was observed. Immunohistochemical staining revealed that over 85% of the neuronal cell bodies in the otic ganglion contained immunoreactivity to VAChT or ChAT. VIP-immunoreactive perikarya comprised approximately 10% of the ganglionic cells. Double staining revealed the presence of VAChT+ and NOS+ neurons which amounted to about 45% of the nerve cells in the otic ganglion. NOS+ only perikarya comprised approx. 15% of all the neurons. Immunoreactivity to enkephalins, substance P, somatostatin, and galanin was expressed in single nerve cell bodies and nerve fibers except numerous substance P+ intraganglionic nerve fibers. Some of them were stained also for CGRP. Single neurons stained for tyroxine hydroxylase. Our results, compared with findings in other rodent species suggest the existence of interspecies differences in the morphology, cellular structure, and immunohistochemical properties of the head autonomic ganglia in mammals.

Voltage to Calcium Transformation Enhances Direction Selectivity in Drosophila T4 Neurons.

An important step in neural information processing is the transformation of membrane voltage into calcium signals leading to transmitter release. However, the effect of voltage to calcium transformation on neural responses to different sensory stimuli is not well understood. Here, we use in vivo two-photon imaging of genetically encoded voltage and calcium indicators, ArcLight and GCaMP6f, respectively, to measure responses in direction-selective T4 neurons of female Drosophila Comparison between ArcLight and GCaMP6f signals reveals calcium signals to have a significantly higher direction selectivity compared with voltage signals. Using these recordings, we build a model which transforms T4 voltage responses into calcium responses. Using a cascade of thresholding, temporal filtering and a stationary nonlinearity, the model reproduces experimentally measured calcium responses across different visual stimuli. These findings provide a mechanistic underpinning of the voltage to calcium transformation and show how this processing step, in addition to synaptic mechanisms on the dendrites of T4 cells, enhances direction selectivity in the output signal of T4 neurons. Measuring the directional tuning of postsynaptic vertical system (VS)-cells with inputs from other cells blocked, we found that, indeed, it matches the one of the calcium signal in presynaptic T4 cells.SIGNIFICANCE STATEMENT The transformation of voltage to calcium influx is an important step in the signaling cascade within a nerve cell. While this process has been intensely studied in the context of transmitter release mechanism, its consequences for information transmission and neural computation are unclear. Here, we measured both membrane voltage and cytosolic calcium levels in direction-selective cells of Drosophila in response to a large set of visual stimuli. We found direction selectivity in the calcium signal to be significantly enhanced compared with membrane voltage through a nonlinear transformation of voltage to calcium. Our findings highlight the importance of an additional step in the signaling cascade for information processing within single nerve cells.

Intracellular simulated biophoton stimulation and transsynaptic signal transmission

The traditional theory holds that the information transmission between nerve cells includes electrical and chemical transmission; however, these known functional features do face some difficulties to explain the fast and efficient information processing and cognitive processes in the brain due to the existing functional limitations of neuronal networks, such as the dendritic and axonal propagation delays as well as the chemical synaptic transmission time delay that have been debated for a long time. We generated three kinds of ultraweak lasers, called as simulated biophotons, with different spectra and intensities to implement intracellular stimulation in a single nerve cell of the hippocampal areas in mouse brain slices combined with intracellular membrane potential recording and biophoton imaging techniques. We found that the simulated biophoton stimulation can lead to transsynaptic biophotonic activities and transmission in the ipsilateral and contralateral projection circuits in the hippocampus. The activity and transmission characteristics were related to the spectra and intensities of the simulated biophotons but not to the levels of membrane potentials before stimulation. These findings present specific characteristics of neural biophoton signal transmission, which may be involved in the mechanisms of processing, encoding, and storage of neural signals.

Chemical Imaging and Analysis of Single Nerve Cells by Secondary Ion Mass Spectrometry Imaging and Cellular Electrochemistry.

A nerve cell is a unit of neuronal communication in the nervous system and is a heterogeneous molecular structure, which is highly mediated to accommodate cellular functions. Understanding the complex regulatory mechanisms of neural communication at the single cell level requires analytical techniques with high sensitivity, specificity, and spatial resolution. Challenging technologies for chemical imaging and analysis of nerve cells will be described in this review. Secondary ion mass spectrometry (SIMS) allows for non-targeted and targeted molecular imaging of nerve cells and synapses at subcellular resolution. Cellular electrochemistry is well-suited for quantifying the amount of reactive chemicals released from living nerve cells. These techniques will also be discussed regarding multimodal imaging approaches that have recently been shown to be advantageous for the understanding of structural and functional relationships in the nervous system. This review aims to provide an insight into the strengths, limitations, and potentials of these technologies for synaptic and neuronal analyses.

Emergence of lump-like solitonic waves in Heimburg-Jackson biomembranes and nerves fractal model.

The aim of this study is to extend the soliton propagation model in biomembranes and nerves constructed by Heimburg and Jackson for the case of fractal dimensions. Our analyses are based on the product-like fractal measure concept introduced by Li and Ostoja-Starzewski in their attempt to explore anisotropic fractal elastic media and electromagnetic fields. The mathematical model presented in the paper is formulated for only a part of a single nerve cell (an axon). The analytical and numerical envelop soliton of this equation are reported. The results obtained prove the emergence of lump-type solitonic waves in nerves and biomembranes. In particular, these waves decay algebraically to the background wave in space direction. This scenario is viewed as a particular class of rational localized waves which are solutions of the integrable Ishimori I equation and the (2 + 1) Kadomtsev-Petviashvili I equation. The effects of fractal dimensions are discussed and were found to be significant to some extents.

Cholinergic and adrenergic innervation of the pancreas in chinchilla (Chinchilla Laniger Molina).

Cholinergic and adrenergic innervation of the pancreas in chinchilla (Chinchilla Laniger Molina) was examined in this study. The pancreas is both an exocrine and endocrine gland with autonomic and sensory innervation presented by the numerous nerve fibers and small agglomerations of nerve cells. Investigations were performed on 16 adult chinchillas of both sexes. The material was collected immediately after death of the animals. Histochemical methods: AChE and SPG were used, in addition to routine technique of single and double immunohistochemical (IHC) staining using whole mount specimens and freezing sections with a thickness of 8 to 12 μm. In the immunofluorescence staining, primary antibodies directed against markers used to identify cholinergic - ChAT and VAChT, and adrenergic - DbH and TH neurons. Secondary antibodies were coupled to Alexa Fluor 488 and Alexa Fluor 555 fluorophores. Histochemical studies (AChE) revealed that chinchilla pancreatic cholinergic innervation consisted of ganglionic neurocytes and numerous nerve fibers. These structures are located in the parenchyma of the exocrine part of the organ in close proximity to blood vessels and are present within the walls of the pancreatic ducts and interstitial connective tissue. A delicate fiber network around the Langerhans islets was also observed. The most numerous cholinergic structures were found in the head and tail, and the least numbers were found in the body of the pancreas. The SPG method revealed that adrenergic fibers form a network in the adventitia of blood vessels, and individual fibers run throughout the pancreatic parenchyma. Moreover, adrenergic nerve fibers were observed around the ganglionic neurocytes. This innervation was similar in all parts of the investigated organ. IHC investigations allowed observations of both the cholinergic and adrenergic activities of autonomic nerve structures. Additionally, using ChAT/DbH double staining, colocalization of these substances was observed in the fibers of the pancreatic parenchyma that passed through the cholinergic ganglia. Colocalization of VAChT and TH was found in nerve fibers of the exocrine part, in the walls of blood vessels, and in individual nerve cells. Colocalization of ChAT/DbH and VAChT/TH was observed in the single nerve cells and in the small (2-3 cell) ganglia. ChAT- and DbH-immunopositive nerve fibers were found in the area of the islets of Langerhans. The results indicate a more intense cholinergic innervation of the chinchilla's pancreas, which is represented by both ganglia and nerve fibers, while adrenergic structures are mainly represented by fibers and only single neurocytes. This arrangement of the investigated structures in this species may imply a major role for hormonal control of exocrine secretion in rodents.

Visual response properties of neurons in the superficial layers of the superior colliculus of awake mouse.

Key points In rodents, including mice, the superior colliculus is the major target of the retina, but its visual response is not well characterized.In the present study, extracellular recordings from single nerve cells in the superficial layers of the superior colliculus were made in awake, head‐restrained mice, and their responses to visual stimuli were measured.It was found that these neurons show brisk, highly sensitive and short latency visual responses, a preference for black over white stimuli, and diverse responses to moving patterns.At least five broad classes can be defined by variation in functional properties among units.The results of the present study demonstrate that eye movements have a measurable impact on visual responses in awake animals and show how they may be mitigated in analyses. The mouse is an increasingly important animal model of visual function in health and disease. In mice, most retinal signals are routed through the superficial layers of the midbrain superior colliculus, and it is well established that much of the visual behaviour of mice relies on activity in the superior colliculus. The functional organization of visual signals in the mouse superior colliculus is, however, not well established in awake animals. We therefore made extracellular recordings from the superficial layers of the superior colliculus in awake mice, while the animals were viewing visual stimuli including flashed spots and drifting gratings. We find that neurons in the superficial layers of the superior colliculus of awake mouse generally show short latency, brisk responses. Receptive fields are usually ‘ON–OFF’ with a preference for black stimuli, and are weakly non‐linear in response to gratings and other forms of luminance modulation. Population responses to drifting gratings are highly contrast sensitive, with a robust response to spatial frequencies above 0.3 cycles degree−1 and temporal frequencies above 15 Hz. The receptive fields are also often speed‐tuned or direction‐selective. Analysis of the response across multiple stimulus dimensions reveals at least five functionally distinct groups of units. We also find that eye movements affect measurements of receptive field properties in awake animals, and show how these may be mitigated in analyses. Qualitatively similar responses were obtained in urethane‐anaesthetized animals, although receptive fields in awake animals had higher contrast sensitivity, shorter visual latency and a stronger response to high temporal frequencies.

A pilot study assessing retinal pathology in psychosis using optical coherence tomography: Choroidal and macular thickness

Mounting evidence supports a genetic-vascular-inflammatory etiology of schizophrenia. The retina provides an indirect assessment of inflammation and degeneration in the brain. In particular, the use of spectral domain optical coherence tomography (SD-OCT) has emerged as a powerful tool for examining single retinal nerve cell layers and the choroid, the vascular layer supplying the outer retina. In this study, choroidal and macular thicknesses were measured in six patients with psychosis with either schizophrenia or bipolar disorder, and in 18 age- and sex-matched healthy controls. Mean choroidal thickness was reduced in psychosis, though not significantly so. There was a statistically significant decrease in macular thickness in psychosis patients predominantly affecting the inner layers of the macula. Significant macular thinning may signal vascular, inflammatory, or degenerative processes that may also be occurring in the brain. This is one of the first studies to examine choroidal thickness in psychosis. Further studies are needed to determine whether the retinal changes in psychosis are correlated with microvascular dysfunction, neuroinflammation, and neurodegeneration.

Electrochemical Measurements of Optogenetically Stimulated Quantal Amine Release from Single Nerve Cell Varicosities in Drosophila Larvae.

The nerve terminals found in the body wall of Drosophila melanogaster larvae are readily accessible to experimental manipulation. We used the light-activated ion channel, channelrhodopsin-2, which is expressed by genetic manipulation in Type II varicosities to study octopamine release in Drosophila. We report the development of a method to measure neurotransmitter release from exocytosis events at individual varicosities in the Drosophila larval system by amperometry. A microelectrode was placed in a region of the muscle containing a varicosity and held at a potential sufficient to oxidize octopamine and the terminal stimulated by blue light. Optical stimulation of Type II boutons evokes exocytosis of octopamine, which is detected through oxidization at the electrode surface. We observe 22700±4200 molecules of octopamine released per vesicle. This system provides a genetically accessible platform to study the regulation of amine release at an intact synapse.

Electrochemical Measurements of Optogenetically Stimulated Quantal Amine Release from Single Nerve Cell Varicosities in Drosophila Larvae

AbstractThe nerve terminals found in the body wall of Drosophila melanogaster larvae are readily accessible to experimental manipulation. We used the light‐activated ion channel, channelrhodopsin‐2, which is expressed by genetic manipulation in Type II varicosities to study octopamine release in Drosophila. We report the development of a method to measure neurotransmitter release from exocytosis events at individual varicosities in the Drosophila larval system by amperometry. A microelectrode was placed in a region of the muscle containing a varicosity and held at a potential sufficient to oxidize octopamine and the terminal stimulated by blue light. Optical stimulation of Type II boutons evokes exocytosis of octopamine, which is detected through oxidization at the electrode surface. We observe 22700±4200 molecules of octopamine released per vesicle. This system provides a genetically accessible platform to study the regulation of amine release at an intact synapse.

Electrophysiological Properties of Ventral Medullary Respiratory Neurons in Rats Submitted to Chronic Intermittent Hypoxia

The central network controlling breathing exhibits plasticity in response to environmental challenges, such as chronic intermittent hypoxia (CIH). For a better understanding of the effects of CIH on the central generation of breathing, we combined single cell qRT-PCR, nerve and whole cell patch clamp recordings of Bötzinger Complex (BötC) and pre-Bötzinger Complex (pre-BötC) respiratory neurons in in situ preparations from control and CIH rats. In the BötC, we recorded 32 augmenting-expiratory (aug-E; glicinergic) and 24 post-inspiratory (post-I; 19 glicinergic and 5 glutamatergic) neurons, which present distinct intrinsic electrophysiological properties. CIH decreased the superior laryngeal nerve (SLN) post-inspiratory activity, by increasing potassium-mediated leak current in the post-I neurons, and increased the duration of expiration by changes in the aug-E neurons. In the pre-BötC, we recorded four types of neurons: pre-inspiratory (pre-I; 15 glutamatergic, 9 GABAergic and 11 glicinergic), early-inspiratory (19 glicinergic neurons), ramp-inspiratory (13 glicinergic and 9 GABAergic) and late-inspiratory (7 GABAergic and 9 glicinergic). CIH enhanced SLN pre-I activity, by increasing the inward riluzole-sensitive persistent sodium current in the intrinsically bursting glutamatergic pre-I neurons. These data show that CIH triggers a reconfiguration of synaptic interaction within the respiratory network in rats, which seems to be an adaptive process required for appropriated pulmonary ventilation and regulation of upper airway resistance. Support: FAPESP, CAPES and CNPQ.

Larry Cohen, indomitable pioneer.

Larry Cohen, indomitable pioneer.

New insights on vertebrate olivo-cerebellar climbing fibers from computerized morphological reconstructions

Characterization of neuronal connectivity is essential to understanding the architecture of the animal nervous system. Specific labeling and imaging techniques can visualize axons and dendrites of single nerve cells. Two-dimensional manual drawing has long been used to describe the morphology of labeled neuronal elements. However, quantitative morphometry, which is essential to understanding functional significance, cannot be readily extracted unless the detailed neuronal geometry is comprehensively reconstructed in three-dimensional space. We have recently applied an accurate and robust digital reconstruction system to cerebellar climbing fibers, which form highly dense and complex terminal arbors as one of the strongest presynaptic endings in the vertebrate nervous system. Resulting statistical analysis has shown how climbing fibers morphology is special in comparison to other axonal terminals. While thick primary branches may convey excitation quickly and faithfully to the far ends, thin tendril branches, which have a larger bouton density, form the majority of presynaptic outputs. This data set, now publicly available from NeuroMorpho.Org for further modeling and analysis, may constitute the first detailed and comprehensive digital reconstruction of the complete axonal terminal field with identified branch types and full accounting of boutons for any neuronal class in the vertebrate brain.

Synthetic hydrogel platform for three-dimensional culture of embryonic stem cell-derived motor neurons.

Culturing mammalian neurons in three-dimensional (3D) microenvironments that more closely recapitulate critical biochemical and biophysical aspects of the developing or adult central nervous system (CNS) milieu remains a significant challenge in neurobiological studies and in regenerative medicine. Here, we aimed to exploit recent advances in poly(ethylene glycol) (PEG) hydrogel chemistries to define a synthetic niche capable of supporting the culture and axonal outgrowth of both aggregated and dissociated mouse embryonic stem cell-derived motor neurons (ESMNs). Using thiol-ene click chemistry to create peptide crosslinked PEG hydrogels, we identified a hydrogel formulation that promotes neuronal survival and axon outgrowth through cell-extracellular matrix interactions, such as those between the laminin-derived peptide YIGSR and its integrin, and that allows neurons to remodel their extracellular environment through matrix metalloproteinase (MMP)-mediated polymer network degradation. Our results demonstrate a 3D platform for culture of both aggregated and single mammalian motor nerve cells that not only permits cell survival over more than a week of culture, but also allows for the robust extension of motor axons. In addition, the optical transparency of the hydrogel allows simultaneous imaging of live cell functions, and as such, this material system should prove useful for studying fundamental aspects of neuronal development.

Swainsonine as a lysosomal toxin affects dopaminergic neurons

Swainsonine (SW) is an indolizidine triol plant alkaloid isolated from the species Astragalus, colloquially termed locoweed. When chronically ingested by livestock and wildlife, symptoms include severe neuronal disturbance. Toxicity to the central and peripheral nervous system is caused by inhibition of lysosomal α-mannosidase (AMA) and accumulation of intracellular oligosaccharide. Consequently, SW has been used as a model substance in investigations of lysosomal storage diseases. Involvement of the basal ganglia has been postulated due to the neuronal symptoms of affected animals. Therefore, primary midbrain cultures from embryonic mice containing dopaminergic neurons were utilized in this study. Neural cells were exposed to SW (0.01-100 μM) for 72 h. AMA activity was 50 % inhibited at 1 μM SW. Cytotoxic changes in cultures were observed above 25 μM SW by increases in lactate dehydrogenase activity and nitric oxide content. Neurotoxicity to dopaminergic cells was visualized by tyrosine hydroxylase immunohistochemistry. Structural degeneration scored as dendritic shortening and shrinkage of cell bodies was dose-dependent and resulted in nerve loss above 25 μM. SW exposure caused progression from reversible to irreversible cytotoxicity. Partial regeneration of AMA-activity in culture was observed on removal of SW. The antioxidative vitamins ascorbic acid and tocopherol (both 100 μM) partially reversed the toxic effect on dopaminergic cells and ascorbic acid decreased AMA inhibition. Thus, neuronal midbrain cell cultures can demonstrate the neurotoxic action of SW and cytoprotective strategies may be tested at a single nerve cell level.

Commentary on “Sulla Fina Anatomia del Cervelletto Umano. Archivio Italiano per le Malatie Nervose 11:90–107.” (English Title: On the Fine Anatomy of the Human Cerebellum)

He stated that [3]: [...] always in the granular layer, with the silver nitrate method, I was able to verify not only the beautiful characteristics of the nerve cell that lay within (characteristics that some have denies) but I was also able to depict their exact dimensions, shapes, positions and ramifications [...]. Before Golgi’s study on cerebellum, there were contradictory ideas on the nature of the granule cell layer. Some histologists regarded the cells contained at this level, as “connective” cells (i.e. glial cells). On the basis of the application of the black reaction, which quite easily allowed a differentiation between nerve and glial cells, Golgi was very determined to affirm that “the so-called granules must be generally considered as small nerve cells provided with three, four, five, and sometimes six processes, usually ramified” and that only one of these processes “should be essentially considered a nerve prolongation” (axon) (Fig. 1). The constant presence of a single ramified nervous prolongation (i.e. of a single axon) for every single nerve cell, later confirmed by Golgi as being applicable to all the regions of the central nervous system, is one of his most enduring contributions to neuroscience. In the external layer of the cerebellar cortex, Golgi discovered bundles of fibres which “run parallel to each other and, in most cases, are of considerable length, many of them running around an entire circumvolution” (Fig. 1). Golgi was able to observe that these fibres, now known as parallel fibres, “could be considered as originating from the granule layer” but was unable to fully trace the axons of the granule cells and so his research failed to reveal that the axons in fact ascend toward the most external region of the cortex and then bifurcates in the shape of a T, giving origin to the parallel fibres. This was one of the first important discoveries of Santiago Ramon y Cajal which was made with the aid of the black reaction in 1888 [8]. Furthermore, Golgi described two kinds of “big nerve cells” in the granular layer—the first were “long and narrow cells irregularly fusiform, with a maximum diameter parallel to the surface of the circumvolution” and the others were “irregularly P. Mazzarello (*) Historical Museum of the University and Department of Experimental Medicine, University of Pavia, Pavia, Italy e-mail: paolo.mazzarello@unipv.it

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian <em>Dugesia japonica</em>

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from these animals reform the original body plan following regeneration of missing body structures. For example if a 'trunk' fragment is cut from an intact worm, a new 'head' will regenerate anteriorly and a 'tail' will regenerate posteriorly restoring the original 'head-to-tail' polarity of body structures prior to amputation (Figure 1A). Regeneration is driven by planarian stem cells, known as 'neoblasts' which differentiate into ~30 different cell types during normal body homeostasis and enforced tissue regeneration. This regenerative process is robust and easy to demonstrate. Owing to the dedication of several pioneering labs, many tools and functional genetic methods have now been optimized for this model system. Consequently, considerable recent progress has been made in understanding and manipulating the molecular events underpinning planarian developmental plasticity2-9. The planarian model system will be of interest to a broad range of scientists. For neuroscientists, the model affords the opportunity to study the regeneration of an entire nervous system, rather than simply the regrowth/repair of single nerve cell process that typically are the focus of study in many established models. Planarians express a plethora of neurotransmitters10, represent an important system for studying evolution of the central nervous system11, 12 and have behavioral screening potential13, 14. Regenerative outcomes are amenable to manipulation by pharmacological and genetic apparoaches. For example, drugs can be screened for effects on regeneration simply by placing body fragments in drug-containing solutions at different time points after amputation. The role of individual genes can be studied using knockdown methods (in vivo RNAi), which can be achieved either through cycles of microinjection or by feeding bacterially-expressed dsRNA constructs8, 9, 15. Both approaches can produce visually striking phenotypes at high penetrance- for example, regeneration of bipolar animals16-21. To facilitate adoption of this model and implementation of such methods, we showcase in this video article protocols for pharmacological and genetic assays (in vivo RNAi by feeding) using the planarian Dugesia japonica.

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian <em>Dugesia japonica</em>

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from these animals reform the original body plan following regeneration of missing body structures. For example if a 'trunk' fragment is cut from an intact worm, a new 'head' will regenerate anteriorly and a 'tail' will regenerate posteriorly restoring the original 'head-to-tail' polarity of body structures prior to amputation (Figure 1A).Regeneration is driven by planarian stem cells, known as 'neoblasts' which differentiate into ~30 different cell types during normal body homeostasis and enforced tissue regeneration. This regenerative process is robust and easy to demonstrate. Owing to the dedication of several pioneering labs, many tools and functional genetic methods have now been optimized for this model system. Consequently, considerable recent progress has been made in understanding and manipulating the molecular events underpinning planarian developmental plasticity2-9.The planarian model system will be of interest to a broad range of scientists. For neuroscientists, the model affords the opportunity to study the regeneration of an entire nervous system, rather than simply the regrowth/repair of single nerve cell process that typically are the focus of study in many established models. Planarians express a plethora of neurotransmitters10, represent an important system for studying evolution of the central nervous system11, 12 and have behavioral screening potential13, 14. Regenerative outcomes are amenable to manipulation by pharmacological and genetic apparoaches. For example, drugs can be screened for effects on regeneration simply by placing body fragments in drug-containing solutions at different time points after amputation. The role of individual genes can be studied using knockdown methods (in vivo RNAi), which can be achieved either through cycles of microinjection or by feeding bacterially-expressed dsRNA constructs8, 9, 15. Both approaches can produce visually striking phenotypes at high penetrance- for example, regeneration of bipolar animals16-21. To facilitate adoption of this model and implementation of such methods, we showcase in this video article protocols for pharmacological and genetic assays (in vivo RNAi by feeding) using the planarian Dugesia japonica.