Pathway specific expression of neuropeptides and autonomic control of the vasculature

In this article, we review the immunohistochemical evidence for the pathway-specific expression of co-existing neuropeptides in autonomic vasomotor neurons, and examine the functional significance of these expression patterns for the autonomic regulation of the vasculature. Most final motor neurons in autonomic vasomotor pathways contain neuropeptides in addition to non-peptide co-transmitters such as catecholamines, acetylcholine and nitric oxide. Neuropeptides also occur in preganglionic vasomotor neurons. The precise combinations of neuropeptides expressed by neurons in vasomotor pathways vary with species, vascular bed, and the level within the vascular bed. This applies to both vasoconstrictor and vasodilator pathways. There is a similar degree of variation in the expression of neuropeptide receptors in the vasculature. Consequently, the contributions of different peptides to autonomic vasomotor control are closely matched to the functional requirements of specific vascular beds. This arrangement allows for a high degree of precision in vascular control in normal conditions and has the potential for considerable plasticity under pathophysiological conditions.     

Polysynaptic subcircuits in the neocortex: spatial and temporal diversity

Inhibitory pathways in the neocortex display a variety of temporal and spatial patterns, maintaining a dynamic balance with excitatory synaptic activity. Recent studies have revealed prevalent polysynaptic subcircuits within the neocortical microcircuitry. These subcircuits involve excitatory and inhibitory connections that are activated by neurons both in supragranular and infragranular cortical layers and mediated by different mechanisms. Interestingly, in these subcircuits inhibition is induced by discharge of pyramidal cells, and excitation is caused by specific types of GABAergic interneurons. The different polysynaptic subcircuits are discussed with respect to their spatial and temporal properties and their possible functional role in cortical processing.     

Axonal ephrinA/EphA interactions, and the emergence of order in topographic projections

In the classical view of axon guidance, neurons send out axons which are endowed with guidance receptors enabling them to find their (distant) target areas by an interaction with their ligands expressed in specific spatio-temporal patterns along their pathways and in their target area. However, this view has recently been confounded by more detailed analyses of, for example, the expression patterns of EphAs and ephrinAs in the retinotectal projection. Here ephrinA 'ligands' are expressed not only in the target area but also on the projecting RGC axons, and EphA 'receptors' not only on retinal ganglion cell (RGC) axons but also in the target area itself. This review describes the on-going functional characterisation of the surprising co-expression of ephrinAs and EphAs on retinal ganglion cell (RGC) axons and other cell types. It also investigates the function of ephrinAs as receptors and describes their interaction with co-receptors involved in mediating this function.     

Whole-Brain Mapping of Inputs to Projection Neurons and Cholinergic Interneurons in the Dorsal Striatum

The dorsal striatum integrates inputs from multiple brain areas to coordinate voluntary movements, associative plasticity, and reinforcement learning. Its projection neurons consist of the GABAergic medium spiny neurons (MSNs) that express dopamine receptor type 1 (D1) or dopamine receptor type 2 (D2). Cholinergic interneurons account for a small portion of striatal neuron populations, but they play important roles in striatal functions by synapsing onto the MSNs and other local interneurons. By combining the modified rabies virus with specific Cre- mouse lines, a recent study mapped the monosynaptic input patterns to MSNs. Because only a small number of extrastriatal neurons were labeled in the prior study, it is important to reexamine the input patterns of MSNs with higher labeling efficiency. Additionally, the whole-brain innervation pattern of cholinergic interneurons remains unknown. Using the rabies virus-based transsynaptic tracing method in this study, we comprehensively charted the brain areas that provide direct inputs to D1-MSNs, D2-MSNs, and cholinergic interneurons in the dorsal striatum. We found that both types of projection neurons and the cholinergic interneurons receive extensive inputs from discrete brain areas in the cortex, thalamus, amygdala, and other subcortical areas, several of which were not reported in the previous study. The MSNs and cholinergic interneurons share largely common inputs from areas outside the striatum. However, innervations within the dorsal striatum represent a significantly larger proportion of total inputs for cholinergic interneurons than for the MSNs. The comprehensive maps of direct inputs to striatal MSNs and cholinergic interneurons shall assist future functional dissection of the striatal circuits.     

Specificity of cortical synaptic connectivity: Emphasis on perspectives gained from quantitative electron microscopy

This report traces the historical development of concepts regarding the specificity of synaptic connectivity in the cerebral cortex as viewed primarily from the perspective of electron microscopy. The occurrence of stereotypical patterns of connection (e.g., contrasting synaptic patterns on the surfaces of spiny vs. non-spiny neurons, the general consistency with which axonal pathways impinge on and originate within specific cortical areas and layers, triadic synaptic relationships) implies that cortical connectivity is highly structured. The high degree of order characterizing many aspects of cortical organization is mirrored by an equally ordered arrangement of synaptic connections between specific types of neurons. This observation is based on quantitative electron microscopic studies of synapses between identified neurons and from the results of correlative anatomical/electrophysiological investigations. The recognition of recurring synaptic patterns and responses between specific neurons has generated increased support for the notion of specificity of synaptic connections at the expense of randomness, but the role of specificity in cortical function is an unresolved question. At the core of cortical processing lie myriad possibilities for computation provided by the wealth of synaptic connections involving each cortical neuron. Specificity, by limiting possibilities for connection, can impose an order on synaptic interactions even as processes of dynamic selection or synaptic remodeling ensure the constant formation and dissolution of cortical circuits. These operations make maximal use of the richness of cortical synaptic connections to produce a highly flexible system, irrespective of the degree of randomness or specificity that obtains for cortical wiring at any particular time.     

Adenovirus-mediated WGA gene delivery for transsynaptic labeling of mouse olfactory pathways

Detailed knowledge of neuronal connectivity patterns is indispensable for studies of various aspects of brain functions. We previously established a genetic strategy for visualization of multisynaptic neural pathways by expressing wheat germ agglutinin (WGA) transgene under the control of neuron type-specific promoter elements in transgenic mice and Drosophila. In this paper, we have developed a WGA-expressing recombinant adenoviral vector system and applied it for analysis of the olfactory system. When the WGA-expressing adenovirus was infused into a mouse nostril, various types of cells throughout the olfactory epithelium were infected and expressed WGA protein robustly. WGA transgene products in the olfactory sensory neurons were anterogradely transported along their axons to the olfactory bulb and transsynaptically transferred in glomeruli to dendrites of the second-order neurons, mitral and tufted cells. WGA protein was further conveyed via the lateral olfactory tract to the olfactory cortical areas including the anterior olfactory nucleus, olfactory tubercle, piriform cortex and lateral entorhinal cortex. In addition, transsynaptic retrograde labeling was observed in cholinergic neurons in the horizontal limb of diagonal band, serotonergic neurons in the median raphe nucleus, and noradrenergic neurons in the locus coeruleus, all of which project centrifugal fibers to the olfactory bulb. Thus, the WGA-expressing adenovirus is a useful and powerful tool for tracing neural pathways and could be used in animals that are not amenable to the transgenic technology.     

Pnn and SR family proteins are differentially expressed in mouse central nervous system

Pinin (pnn) is an SR-related protein that is ubiquitously expressed in most cell types and functions in regulating pre-mRNA splicing and mRNA export. Previously, we demonstrated that pnn is expressed in all tissues during mouse embryonic development with highest levels of expression in the central nervous system (CNS). Here we show that pnn and other SR proteins including SC35 are differentially expressed in the adult mouse CNS, displaying cell type-specific distribution patterns. Immunohistochemical analysis of whole-brain sections showed that levels of pnn and SR proteins expression were very low or nonexistent in the corpus callosum and white matter of cerebellum and spinal cord. Double-immunostaining with antibodies specific to neuron or glial cells showed that most astrocytes and microglia expressed neither pnn nor SR proteins. In contrast, oligodendrocytes and neurons expressed moderate and high levels, respectively, of both pnn and SR proteins. These results suggest that astrocytes are unique among cell types of neuroblast origin in terms of expression SR family proteins. Our results pave the way for future studies of the functional roles of pnn and SR family proteins in adults.     

Motor Axon Pathfinding

Motor neurons are functionally related, but represent a diverse collection of cells that show strict preferences for specific axon pathways during embryonic development. In this article, we describe the ligands and receptors that guide motor axons as they extend toward their peripheral muscle targets. Motor neurons share similar guidance molecules with many other neuronal types, thus one challenge in the field of axon guidance has been to understand how the vast complexity of brain connections can be established with a relatively small number of factors. In the context of motor guidance, we highlight some of the temporal and spatial mechanisms used to optimize the fidelity of pathfinding and increase the functional diversity of the signaling proteins.Motor neurons residing in the brain stem and spinal cord extend axons into the periphery and are the final relay cells for locomotor commands. These cells are among the longest projection neurons in the body and their axons follow stereotypical pathways during embryogenesis to synapse with muscle and sympathetic/parasympathetic targets. Cellular studies of motor axon navigation in developing chick and zebrafish embryos have shown that motor neurons located at different rostrocaudal positions show specific preferences for axonal pathways (see Landmesser 2001; Lewis and Eisen 2003 for reviews). This early cellular research laid the foundation for molecular studies of motor axon guidance by establishing the concept that motor neurons are in fact a diverse cell population. The molecular studies covered in this article have sought to identify genetic differences between motor neurons and to characterize the signaling pathways that underlie the specificity of motor axon targeting.     

Proteome analysis of primary neurons and astrocytes from rat cerebellum

Neurons and astrocytes are predominant cell types in brain and have distinguished morphological and functional features. Although several proteomics studies were carried out on the brain, work on individual brain cells is limited. Generating individual proteomes of neurons and astrocytes, however, is mandatory to assign protein expression to cell types rather than to tissues. We aimed to provide maps of rat primary neurons and astrocytes using two-dimensional gel electrophoresis with subsequent in-gel digestion, followed by MALDI-TOF/TOF. 428 protein spots corresponding to 226 individual proteins in neurons and 406 protein spots representing 228 proteins in astrocytes were unambiguously identified. Proteome data include proteins from several cascades differentially expressed in neurons and astrocytes, and specific expressional patterns of antioxidant, signaling, chaperone, cytoskeleton, nucleic acid binding, proteasomal, and metabolic proteins are demonstrated. We herein present a reference database of primary rat primary neuron and astrocyte proteomes and provide an analytical tool for these structures. The concomitant expressional patterns of several protein classes are given and potential neuronal and astrocytic marker candidates are presented.     

Correlation of extracellular matrix components with the cytoarchitecture of mouse Peyer's patches

Summary The distribution patterns of extracellular matrix elements were determined to ascertain whether they play a role in the localization of lymphocytes in discrete T-cell, B-cell and dome antigen-processing domains within Peyer's patches. Antibodies against collagen types I, III and IV, laminin and fibronectin were applied to cryosections of mouse Peyer's patches and localized by direct or indirect immunoperoxidase methods. T-cell domains were identified with a monoclonal antibody against Thy-1.2. Labeled reticular fibers in distinctive patterns were more numerous in parafollicular and dome areas than within follicles. Germinal centers contained few such fibers. In parafollicular areas, fibers were oriented predominantly toward follicle domes; their distribution corresponded to T-cell zones and lymphocyte traffic areas, with their orientation being parallel to the migration pathways of lymphocytes from high endothelial venules to the antigen-processing domes. Subepithelial and subendothelial basal laminae were immunopositive for type-IV collagen, laminin and fibronectin. The dome subepithelial basal lamina had pore-like discontinuities through which lymphocytes migrated to and from the epithelium. The correspondence of the distribution patterns of extracellular matrix to specific functional domains of Peyer's patches suggests that this matrix provides a structural framework for lymphocyte migration and localization.     

Temporal-to-spatial dynamic mapping,flexible recognition,and temporal correlations in an olfactory cortex model

 This paper proposes temporal-to-spatial dynamic mapping inspired by neural dynamics of the olfactory cortex. In our model the temporal structure of olfactory-bulb patterns is mapped to the spatial dynamics of the ensemble of cortical neurons. This mapping is based on the following biological mechanism: while anterior part of piriform cortex can be excited by the afferent input alone, the posterior areas require both afferent and association signals, which are temporally correlated in a specific way. One of the functional types of the neurons in our model corresponds to the cortical spatial dynamics and encodes odor components, and another represents temporal activity of association-fiber signals, which, we suggest, may be relevant to the encoding of odor concentrations. The temporal-to-spatial mapping and distributed representation of the model enable simultaneous rough cluster classification and fine recognition of patterns within a cluster as parts of the same dynamic process. The model is able to extract and segment the components of complex odor patterns which are spatiotemporal sequences of neural activity. Received: 16 October 2001 / Accepted in revised form: 7 February 2002     

Specific gene transfer to neurons, endothelial cells and hematopoietic progenitors with lentiviral vectors

We present a flexible and highly specific targeting method for lentiviral vectors based on single-chain antibodies recognizing cell-surface antigens. We generated lentiviral vectors specific for human CD105(+) endothelial cells, human CD133(+) hematopoietic progenitors and mouse GluA-expressing neurons. Lentiviral vectors specific for CD105 or for CD20 transduced their target cells as efficiently as VSV-G pseudotyped vectors but discriminated between endothelial cells and lymphocytes in mixed cultures. CD133-targeted vectors transduced CD133(+) cultured hematopoietic progenitor cells more efficiently than VSV-G pseudotyped vectors, resulting in stable long-term transduction. Lentiviral vectors targeted to the glutamate receptor subunits GluA2 and GluA4 exhibited more than 94% specificity for neurons in cerebellar cultures and when injected into the adult mouse brain. We observed neuron-specific gene modification upon transfer of the Cre recombinase gene into the hippocampus of reporter mice. This approach allowed targeted gene transfer to many cell types of interest with an unprecedented degree of specificity.     

Mouse transgenesis in a single locus with independent regulation for multiple fluorophores

A major barrier to complex experimental design in mouse genetics is the allele problem: combining three or more alleles is time-consuming and inefficient. Here, we solve this problem for transgenic animals with a simple modification of existing BAC transgenesis protocols, and generate triple-colored 'prism' mice in which the major cell types of the brain: neurons, astrocytes, and oligodendrocytes, are each labeled with a distinct fluorophore. All three fluorophores are expressed from the same locus, yet each fluorophore is expressed in an independent temporal and spatial pattern. All three transgenes are generally co-inherited across multiple generations with stable genomic copy number and expression patterns. This generic solution should permit more sophisticated experimental manipulations to assess functional interactions amongst populations of cell types in vivo in a more rapid and efficient manner.     

Tissue-Specific Functional Networks for Prioritizing Phenotype and Disease Genes

Integrated analyses of functional genomics data have enormous potential for identifying phenotype-associated genes. Tissue-specificity is an important aspect of many genetic diseases, reflecting the potentially different roles of proteins and pathways in diverse cell lineages. Accounting for tissue specificity in global integration of functional genomics data is challenging, as “functionality” and “functional relationships” are often not resolved for specific tissue types. We address this challenge by generating tissue-specific functional networks, which can effectively represent the diversity of protein function for more accurate identification of phenotype-associated genes in the laboratory mouse. Specifically, we created 107 tissue-specific functional relationship networks through integration of genomic data utilizing knowledge of tissue-specific gene expression patterns. Cross-network comparison revealed significantly changed genes enriched for functions related to specific tissue development. We then utilized these tissue-specific networks to predict genes associated with different phenotypes. Our results demonstrate that prediction performance is significantly improved through using the tissue-specific networks as compared to the global functional network. We used a testis-specific functional relationship network to predict genes associated with male fertility and spermatogenesis phenotypes, and experimentally confirmed one top prediction, Mbyl1. We then focused on a less-common genetic disease, ataxia, and identified candidates uniquely predicted by the cerebellum network, which are supported by both literature and experimental evidence. Our systems-level, tissue-specific scheme advances over traditional global integration and analyses and establishes a prototype to address the tissue-specific effects of genetic perturbations, diseases and drugs.