Combining computational prediction of cis-regulatory elements with a new enhancer assay to efficiently label neuronal structures in the medaka fish

The developing vertebrate nervous system contains a remarkable array of neural cells organized into complex, evolutionarily conserved structures. The labeling of living cells in these structures is key for the understanding of brain development and function, yet the generation of stable lines expressing reporter genes in specific spatio-temporal patterns remains a limiting step. In this study we present a fast and reliable pipeline to efficiently generate a set of stable lines expressing a reporter gene in multiple neuronal structures in the developing nervous system in medaka. The pipeline combines both the accurate computational genome-wide prediction of neuronal specific cis-regulatory modules (CRMs) and a newly developed experimental setup to rapidly obtain transgenic lines in a cost-effective and highly reproducible manner. 95% of the CRMs tested in our experimental setup show enhancer activity in various and numerous neuronal structures belonging to all major brain subdivisions. This pipeline represents a significant step towards the dissection of embryonic neuronal development in vertebrates.     

Dispersion of the neurons expressing layer specific markers in the reeler brain

Neurons with similar functions including neuronal connectivity and gene expression form discrete condensed structures within the vertebrate brain. This is exemplified within the circuitry formed by the cortical layers and the neuronal nuclei. It is well known that the Reelin protein is required for development of these neuronal structures in rodents and human, but the function of Reelin remains controversial. In this report, we used “layer‐specific markers” of the cerebral cortex to carry out detailed observations of spatial distribution of the neuronal subpopulations in the brain of the Reelin deficient mouse, reeler. We observed a spatially dispersed expression of the markers in the reeler cerebral cortex. These markers are expressed also in other laminated and non‐laminated structures of brain, in which we observed similar abnormal gene expression. Our observations suggest that neurons within the brain structures (such as the layers and the nuclei), which normally exhibit condensed distribution of marker expressions, loosen their segregation or scatter by a lack of Reelin.     

Study of cardio- and hemodynamic reactions to administration of GABA into the neuronal structures of the ventrolateral area of the medulla oblongata of cats

The data obtained in acute experiments on cats have indicated that the most GABA-sensitive injected neuronal structures in the ventrolateral medulla appear to be located in the vicinity to the roots of the XII cranial nerves. GABA injected into these structures just rostrally to the roots induced hypotensive reactions due to heart rate and myocardial contractile activity decrease, besides inhibition of sympathetic vasoconstrictor influences. GABA injected into the neuronal structures in the caudal ventrolateral medulla induced hypertensive reactions, to a large extent due to increase in heart rate and contractile activity of myocardium.     

Effects of experimental factors on the tinctorial properties of structural elements of the mammalian cerebral cortex (intravital microscopic study)]

Effects of a vital dye--methylene blue--on brain neuronal structures were investigated in the alive preparations of mammal brains by means of contact optical microscope in falling polarization light. Tinctorial properties of neuronal cortical structures were studied under the influence of a number of experimental factors (illumination conditions, dye concentration, temperature regime and oxygen supply to the preparation). A certain dependence in the character of dyeing and bleaching neuronal cells on the factors mentioned above was stated. Conditions for appearance of the alternating photodynamical effect and measures to prevent it were clarified. Practical recommendations were given for application methylene blue in prevital morphological investigations of mammal central nervous system.     

An analysis of the neuronal processes in the activating brain systems of the rat during the acquisition of an instrumental defense reflex

In chronic experiment, a defensive conditioned reflex was elaborated in rats with electrodes implanted in the reticular oral pons nucleus, cortico-medial group of amygdalar nuclei, ventromedial hypothalamic nucleus and central grey substance of the midbrain. Synchronization of the activity of neuronal groups of emotiogenic formations in the studied brain structures, became enhanced at formation of the conditioned reflex. A dependence was revealed of the level of correlation of limbic neuronal groups activity on functional state of the reticular formation, as well as neurochemical correlation mechanisms. The increase (decrease) of coefficients of correlation of the activity of neuronal ensembles of limbic structures was accompanied by a change of evoked potentials parameters recorded in them.     

Sensory endings in the intramural ganglia of the stomach. Light optical and electron microscopic study

Puppies and young dogs at the age of 3 days up to 2 months were used in the investigation. In total stomach preparations neuronal structures were stained with methylene blue after A. S. Dogel. Routine electron microscopic studies were carried out. Within the structure of intramuscular vegetative plexus of the stomach a sensory plexus was demonstrated, its terminals forming receptive bushy structures in all the tissues. Especially numerous the sensory endings were in neuronal cords and ganglia. The sensory endings innervated all the elements in ganglia. Ultrastructure of receptor terminals situated in the intramural ganglia of the stomach corresponded to that of terminals in tissue mechanoreceptors described for skin, auriculum, carotid sinus and trachea. Peculiar multilayer lamillar structures formed by cytoplasmic axonal processes of lemmocytes around lance-shaped, arranged in rosary fashion receptor varicosities were described. Contact of receptor terminals with the neuronal cell body was demonstrated. Functional nature of the tissue receptors described was discussed.     

Loss of nervous system complexity – Morphological analyses shed light on the neuronal evolution in Myzostomida (Annelida)

Myzostomida are putatively nested within the errant Annelida and exhibit a unique morphology. The latter fact might possibly be related to their long host-dependent radiation. Hence, an incomplete segmentation, lack of prominent sensory structures in adults and a dorso-ventrally flattened body are just some examples. Although numerous investigations of the nervous system exist for myzostomids, detailed ultrastructural as well as histological examinations of neuronal structures are lacking so far. Therefore, we investigate the nervous system of Myzostoma cirriferum Leuckart, 1836 using a comparative approach including paraffin histology, serial semi-thin sections, immunohistochemistry and ultrastructural investigations. Our analyses reveal a lack of complexity within the anterior-most neuronal condensation (herein called brain) of adult specimens. Hence, prominent tracts or glomeruli are absent, and a glial layer surrounding the brain or radial-like glial cells are lacking. Nevertheless, the ultrastructure of the ventral nervous system is comparable to other Errantia. Therefore, our investigations hint towards a reduction of neuronal complexity in Myzostomida. Additionally, an ontogenetic simplification seems plausible, although further investigations are necessary to verify such a hypothesis. A simplification of neuronal structures due to a drastic change in lifestyle was so far mainly observed for basally branching annelid clades.     

The Role of the Neuronal Network in Functional Evolution of the Mammalian Telencephalon

During evolution of the vertebrate telencephalon the analyzing-synthesizing function was divided between two structures: the screen one, optimal for mechanisms of discrete analysis of information, and the neuronal network, in which cortical, functionally specialized modules can create the generalized equivalent of their activity. In the screen and reticular structures, each of these mechanisms got a possibility of developing independently without restricting functional capabilities of the other one. This resulted in formation of two telencephalon structures developing in parallel and functionally related, the cortex and the neostriatum. Experimental data indicate that the neuronal network of neostriatum is a field for interaction of corticofugal signals. These signals form neuronal mosaics reflecting the dynamics of cortical activity as combination patterns. Thereby, in neostriatum, corticofugal signals spread over a large surface of the cortex are transformed into their three-dimensional equivalent similar to population coding of information that takes place in the brain sensory structures. The established system of interrelationships between the cortex and the neostriatum turned out to be rather universal and economic. As a result, a broad spectrum of functional brain capabilities that we can see in various representatives of mammals was formed during a relatively short time with the minimum of structural changes, mainly quantitative in character.     

Degeneration of neuronal processes after infection with pathogenic, but not attenuated, rabies viruses

The structural alterations of neuronal processes in mice were investigated after the mice were infected with rabies virus (RV). Silver staining of infected brain sections showed severe destruction and disorganization of neuronal processes in mice infected with pathogenic RV but not with attenuated RV. However, neuronal bodies showed very little pathological changes. Electron microscopy revealed the disappearance of intracellular organelles, as well as the disappearance of synaptic structures and vesicles. Infection of primary neurons with pathogenic, but not attenuated, RV resulted in the destruction of neuronal processes and disappearance of microtubule-associated protein 2 and neurofilament immunoreactivity, which suggests that pathogenic RV causes degeneration of neuronal processes possibly by interrupting cytoskeletal integrity.     

Neurochemical mechanisms of the septum in food getting conditioned responses in rats]

The influence of microinjections of monoamines and of glutamic acid into the lateral zone of the septum on a food-procuring conditioned reaction was studied in experiments on rats. Dopamine does not change, while noradrenaline, serotonin and glutamic acid increase the magnitude and number of conditioned food-procuring movements and substantially lengthen the reflex latency. The effects of serotonin are achieved through D-serotonin-, and of noradrenaline, through L-adrenoreactive structures of the neuronal systems of the septum. The influence of glutamic acid on the rat food-procuring conditioned reactions is not linked with the action on the serotonin- or adrenoreactive structures of the septum neuronal systems.     

Equivalence transformations for dendritic Y-junctions: a new definition of dendritic sub-unit

A sequence of equivalence transformations is used to represent the mathematical model of a simply branched neuron with non-homogeneous membrane properties and non-uniform geometry by an entirely equivalent model of an unbranched structure. The analysis indicates how neuronal morphology, in combination with its biophysical properties, shapes neuronal output in response to current input. The equivalence transformations described here reveal the types of operations that are likely to feature in the analysis of complex multi-branched structures, neuronal or otherwise. These transformations provide a new definition of dendritic sub-unit and a basis of a mechanism for characterising local and non-local signal processing within dendritic structures. It is anticipated that the capacity to transform biological neurons into an equivalent unbranched structure will make an important contribution to the understanding of the functional role of neuron geometry as well as to the construction of silicon neurons with realistic biological properties.     

Systems analysis of the integrative activity of the neuron (1974)

This article is aimed at revising the traditional concept of neuronal activity based on pre-eminence of transmembrane potentials and "electric summation" on the neuron surface. It presents a historical survey of the emergence of the prevailing concept on propagation of potentials along conductive structures and reveals the psychological situation that determined the transfer of this concept to dendrites and the neuronal soma. Structural and biophysical properties of the neuron which do not permit information propagation along the neuronal membrane without crude distortion are critically discussed in detail.     

Atypical nucleosome spacing of rat neuronal identifier elements in non-neuronal chromatin.

Rat neuronal identifier (ID) elements are located in chromatin regions that are organized in nucleosomal structures in both neuronal and non-neuronal cells. A subpopulation of ID sequences in chromatin of liver and kidney cells are relatively resistant to micrococcal nuclease digestion and are organized in nucleosomes exhibiting an atypically short repeat length. Other repetitive elements do not show this organization.     

Effects of hyperglycemic and normoglycemic cerebral ischemia on phosphorylation of c-jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK).

Transient global cerebral ischemia leads to delayed neuronal cell death in the hippocampal CA1, caudate putamen and neocortex. If preischemic hyperglycemia exists, the same duration of ischemia recruits additional brain structures, such as dentate gyrus to become damaged. The objective of the present study is to determine whether activation of mitogen-activated protein kinases (MAPKs) plays a role in hyperglycemia-mediated ischemic neuronal damage. Using phopho-specific antibodies against c-jun NH2-terminal kinase (JNK) and p38 MAPK, we studied activation of these two MAPKs in ischemia-vulnerable neocortex and ischemia-resistant dentate gyrus in rats subjected to 15 min of forebrain ischemia and followed by 0.5, 1 and 3 hr of recirculation under normo- and hyperglycemic conditions. The results showed that levels of phosphorylated JNK increased in both normo- and hyperglycemic brains following blood reperfusion for 0.5 hr and persisted up to 3 hr in the neocortex but not in the dentate gyrus, implying JNK may play a role in mediating neuronal cell death after ischemia. However, since hyperglycemia did not further increase phospho-JNK, JNK may not contribute to the detrimental effect of hyperglycemia on neuronal cell death. The amount of phospho-p38 was not altered by ischemia under both normo- and hyperglycemic conditions, suggesting that p38 MAPK may not play a major role in mediating neuronal damage in these two structures.     

Convergence of Signals and Reorganization of Neuronal Activity in a Model of Neuronal Network and in Striatum of the Monkey Brain

To elucidate principles of neuronal organization providing preservation of informational content of converging impulse flows in afferent impulsation of neurons, a comparison is performed of results obtained in the previously carried out experiments on a model of neuronal network and in a study of correlates of behavior in the neuronal network of the monkey brain neostriatum (putamen). This comparison has shown that responses of the neuronal network model to different ratio of input impulse flows and changes of the neostriatal neuronal activity, which accompany different behavioral actions, are seen the most clearly in reorganization of composition of the most active neurons. Each combination of input signals and each behavioral action of the animal correspond to a non-repeated mosaic of neuronal activity. The data obtained indicate that the neuronal network, both real and in the simplest model variant, is able to transform the converging input signals into the mosaic equivalent to their entire combination and thereby to transmit the result of generalization of the input signals of the network to the innervated brain structures.     

Histofluorescent labelling of catecholaminergic structures in rotifers (Aschelminthes) in whole animals

1. Catecholaminergic neuronal structures were investigated in the rotifers Brachionus plicatilis, Asplanchna priodonta and Asplanchna herricki, using three different aqueous histofluorescent methods. 2. The adrenergic receptors were labelled using the dansyl analog of propranolol, a beta-adrenergic blocker. Catecholamine neurotransmitters were visualized by derivatizing with glyoxylic acid and formaldehyde respectively. 3. Although all three methods lead to similar results, dansyl-propranolol gave the most rapid and strong fluorescence. 4. The results reveal a complex and highly developed catecholaminergic neuronal system in all adult organs and sensory structures. While developed embryos in the egg show strong fluorescence, immature eggs do not.     

Rayburst sampling, an algorithm for automated three-dimensional shape analysis from laser scanning microscopy images

Precise quantification of complex three-dimensional (3D) structures from laser scanning microscopy (LSM) images is increasingly necessary in understanding normal function and pathologic processes in biology. This protocol describes a versatile shape analysis algorithm, Rayburst sampling, that generates automated 3D measurements from LSM images. Rayburst defines and efficiently casts a multidirectional core of rays from an interior point to the surface of a solid, allowing precise quantification of anisotropic and irregularly shaped 3D structures. Quantization error owing to the finite voxel representation in digital images is minimized by interpolating intensity values continuously between voxels. The Rayburst algorithm provides a primitive for the development of higher level algorithms that solve specific shape analysis problems. Examples are provided of applications to 3D neuronal morphometry: (i) estimation of diameters in tubular neuronal dendritic branching structures, and (ii) measurement of volumes and surface areas for dendritic spines and spatially complex histopathologic structures.     

Ultrastructural localization of acetylcholinesterase in the cerebellar cortex with special reference to the intersynaptic organelles

Summary Ultrastructural investigation of the synapse indicates that intersynaptic organelles connect prewith post-synaptic structure. These elements may provide a functional continuity between neighbouring neuronal structures, divided from each other under resting conditions.Electron microscipic localization of AChE revealed the exact sites of this enzyme in the cerebellum. AChE located on neuronal surface membranes and on the intersynaptic organelles is obviously active in the hydrolysis of ACh.     

Neuronal interactions between neuropeptide Y (NPY) and catecholaminergic systems in the rat arcuate nucleus as shown by dual immunocytochemistry

Several recent studies have suggested interactions between catecholamine (CA) and neuropeptide Y (NPY) neuronal systems in the rat brain. In order to obtain morphological evidence for such CA/NPY interactions in the arcuate nucleus, we have used a double immunostaining procedure using an anti-tyrosine hydroxylase (TH) antiserum as a marker for catecholamine neurons and an anti-NPY antiserum. This double staining, where the first staining is silver-gold intensified, was detectable at both light and electron microscopic levels. In semi-thin sections, a substantial overlap and close proximity of TH-immunopositive neurons and NPY neuronal elements could be seen within the arcuate nucleus. At the electron microscopic level, direct appositions between TH- and NPY-immunoreactive structures could be detected. These appositions were of axosomatic, axodendritic or axoaxonic types without any synaptic membrane differentiation. Moreover, direct appositions between NPY-immunoreactive structures have also been observed. This morphological study showing appositions between TH and NPY neuronal systems suggest direct interactions between these two systems in the arcuate nucleus.     

Analysis of putative high-mobility-group (HMG) proteins in neuronal and glial nuclei from rabbit brain

Putative high-mobility-group (HMG) proteins 1, 2, and 17 were detected in neuronal and glial nuclei isolated from the cerebral hemisphere of rabbit brain. Although divergent chromatin structures are present in these two populations of brain nuclei (i.e., neuronal nuclei exhibit a short DNA repeat length), no differences were apparent in the electrophoretic mobilities of putative HMG proteins 1, 2, and 17 on SDS gels.