beta -Neuregulin-1 is required for the in vivo development of functional Ca2+-activated K+ channels in parasympathetic neurons.

The development of functional Ca(2+)-activated K(+) channels (K(Ca)) in chick ciliary ganglion (CG) neurons requires interactions with afferent preganglionic nerve terminals. Here we show that the essential preganglionic differentiation factor is an isoform of beta-neuregulin-1. beta-Neuregulin-1 transcripts are expressed in the midbrain preganglionic Edinger-Westphal nucleus at developmental stages that coincide with or precede the normal onset of macroscopic K(Ca) in CG neurons. Injection of beta-neuregulin-1 peptide into the brains of developing embryos evoked a robust stimulation of functional K(Ca) channels at stages before the normal appearance of these channels in CG neurons developing in vivo. Conversely, injection of a neutralizing antiserum specific for beta-neuregulin-1 inhibited the development of K(Ca) channels in CG neurons. Low concentrations of beta-neuregulin-1 evoked a robust increase in whole-cell K(Ca) in CG neurons cocultured with iris target tissues. By contrast, culturing CG neurons with iris cells or low concentrations of beta-neuregulin-1 by themselves was insufficient to stimulate K(Ca). These data suggest that the preganglionic factor required for the development of K(Ca) in ciliary ganglion neurons is an isoform of beta-neuregulin-1, and that this factor acts in concert with target-derived trophic molecules to regulate the differentiation of excitability.

Disruption of gastrulation and heparan sulfate biosynthesis in EXT1-deficient mice.

Mutations in the EXT1 gene are responsible for human hereditary multiple exostosis type 1. The Drosophila EXT1 homologue, tout-velu, regulates Hedgehog diffusion and signaling, which play an important role in tissue patterning during both invertebrate and vertebrate development. The EXT1 protein is also required for the biosynthesis of heparan sulfate glycosaminoglycans that bind Hedgehog. In this study, we generated EXT1-deficient mice by gene targeting. EXT1 homozygous mutants fail to gastrulate and generally lack organized mesoderm and extraembryonic tissues, resulting in smaller embryos compared to normal littermates. RT-PCR analysis of markers for visceral endoderm and mesoderm development indicates the delayed and abnormal development of both of these tissues. Immunohistochemical staining revealed a visceral endoderm pattern of Indian hedgehog (Ihh) in wild-type E6.5 embryos. However, in both EXT1-deficient embryos and wild-type embryos treated with heparitinase I, Ihh failed to associate with the cells. The effect of the EXT1 deletion on heparan sulfate formation was tested by HPLC and cellular glycosyltransferase activity assays. Heparan sulfate synthesis was abolished in EXT1 -/- ES cells and decreased to less than 50% in +/- cell lines. These results indicate that EXT1 is essential for both gastrulation and heparan sulfate biosynthesis in early embryonic development.

Evolution of odorant receptors.

Odorant receptors (ORs) located in the nasal epithelium, at the ciliated surface of olfactory sensory neurons, represent the initial step of a transduction cascade that leads to odor detection. ORs form the largest and most diverse family of G-protein-coupled receptors (GPCRs). They are encoded by a multigene family that has been partially characterized in cyclostomes, teleosts, amphibia, birds and mammals, as well as in Drosophila melanogaster and the nematode Caenorhabditis elegans. As new sequence data emerge, it is increasingly clear that OR primary structure can vary dramatically across phyla. Some chemoreceptors are encoded by genes with little sequence similarity to the prototypical ORs originally isolated in mammals. A large number of sequences are now available allowing a detailed study of the evolutionary implications of OR diversity across species. This review discusses the evolutionary implications of the divergent primary structures of chemoreceptors with identical functions.

Localization of glutamate and glutamate transporters in the sensory neurons of Aplysia.

The sensorimotor synapse of Aplysia has been used extensively to study the cellular and molecular basis for learning and memory. Recent physiologic studies suggest that glutamate may be the excitatory neurotransmitter used by the sensory neurons (Dale and Kandel [1993] Proc Natl Acad Sci USA. 90:7163-7167; Armitage and Siegelbaum [1998] J Neurosci. 18:8770-8779). We further investigated the hypothesis that glutamate is the excitatory neurotransmitter at this synapse. The somata of sensory neurons in the pleural ganglia showed strong glutamate immunoreactivity. Very intense glutamate immunoreactivity was present in fibers within the neuropil and pleural-pedal connective. Localization of amino acids metabolically related to glutamate was also investigated. Moderate aspartate and glutamine immunoreactivity was present in somata of sensory neurons, but only weak labeling for aspartate and glutamine was present in the neuropil or pleural-pedal connective. In cultured sensory neurons, glutamate immunoreactivity was strong in the somata and processes and was very intense in varicosities; consistent with localization of glutamate in sensory neurons in the intact pleural-pedal ganglion. Cultured sensory neurons showed only weak labeling for aspartate and glutamine. Little or no gamma-aminobutyric acid or glycine immunoreactivity was observed in the pleural-pedal ganglia or in cultured sensory neurons. To further test the hypothesis that the sensory neurons use glutamate as a transmitter, in situ hybridization was performed by using a partial cDNA clone of a putative Aplysia high-affinity glutamate transporter. The sensory neurons, as well as a subset of glia, expressed this mRNA. Known glutamatergic motor neurons B3 and B6 of the buccal ganglion also appeared to express this mRNA. These results, in addition to previous physiological studies (Dale and Kandel [1993] Proc Natl Acad Sci USA. 90:7163-7167; Trudeau and Castellucci [1993] J Neurophysiol. 70:1221-1230; Armitage and Siegelbaum [1998] J Neurosci. 18:8770-8779)) establish glutamate as an excitatory neurotransmitter of the sensorimotor synapse.

Odorant receptors: a plethora of G-protein-coupled receptors.

Odorant receptors (ORs) comprise the largest family of G-protein-coupled receptors (GPCRs). They are located in the nasal epithelium, at the ciliated surface of olfactory sensory neurones, where the initial steps of the olfactory transduction cascade occur. ORs are encoded by a large and diverse multi-gene family, which has been characterized in cyclostomes, teleosts, amphibia, birds and mammals, as well as in Drosophila and Caenorhabditis elegans. Here, the range of diversity in OR and chemoreceptor structure is examined, noting that their functions are fundamentally similar to those of many neurotransmitter or neurohormone receptors. It is argued that ORs have emerged directly from other GPCRs independently in many species. According to this view, there is no structural prerequisite for OR identity and any GPCR has the potential to be or become an OR at a given point in evolution.

Regulation of neuronal K(+) currents by target-derived factors: opposing actions of two different isoforms of TGFbeta.

The developmental expression of macroscopic Ca(2+)-activated K(+) currents in chick ciliary ganglion neurons is dependent on an avian ortholog of TGFbeta1, known as TGFbeta4, secreted from target tissues in the eye. Here we report that a different isoform, TGFbeta3, is also expressed in a target tissue of ciliary ganglion neurons. Application of TGFbeta3 inhibits the functional expression of whole-cell Ca(2+)-activated K(+) currents evoked by 12 hour treatment with either TGFbeta1 or beta-neuregulin-1 in ciliary ganglion neurons developing in vitro. TGFbeta3 had no effect on voltage-activated Ca(2+) currents. A neutralizing antiserum specific for TGFbeta3 potentiates stimulation of Ca(2+)-activated K(+) currents evoked by a target tissue (iris) extract in cultured ciliary ganglion neurons, indicating that TGFbeta3 is an inhibitory component of these extracts. Intraocular injection of TGFbeta3 causes a modest but significant inhibition of the expression of Ca(2+)-activated K(+) currents in ciliary ganglion neurons developing in vivo. Further, intraocular injection of a TGFbeta3-neutralizing antiserum stimulates expression of Ca(2+)-activated K(+) currents in ciliary ganglion neurons developing in vivo, indicating that endogenous TGFbeta3 regulates the functional expression of this current. The normal developmental expression of functional Ca(2+)-activated K(+) currents in ciliary ganglion neurons developing in vivo is therefore regulated by two different target-derived isoforms of TGFbeta, which produce opposing effects on the electrophysiological differentiation of these neurons.

A novel family of ancient vertebrate odorant receptors.

Vertebrate odorant receptor (OR) genes have been isolated and characterized in several taxa, including bony fish and mammals. However, the search for more ancient vertebrate OR genes has been unsuccessful to date, indicating that these ancient genes share little sequence identity with previously isolated ORs. The lamprey (Lampetra fluviatilis) olfactory epithelium does not appear to express any of the modern vertebrate ORs previously identified in bony fish and mammals. We have isolated and characterized an ancient family of vertebrate membrane receptors from the olfactory epithelium of the lamprey. Sequence analysis reveals similarities with other Class A (rhodopsin-like) G protein-coupled receptors such as serotonin, dopamine, and histamine receptors, but the expression patterns of members of the new family, as well as certain conserved motifs, strongly suggest that the sequences encode ORs. Sequence similarity within the lamprey OR family is low, and Southern blot analysis suggests reduced-sized subfamilies. This novel vertebrate OR gene family, the most ancient isolated to date, is proposed to be involved in the detection of water-borne molecules in jawless fishes. Lamprey OR genes therefore represent a new level of diversity within the vertebrate OR gene family, but also provide clues as to how vertebrate ORs might have emerged.

Arachidonic acid-sensitive A-currents and multiple Kv4 transcripts are expressed in chick ciliary ganglion neurons.

A-currents (IA) of chick ciliary ganglion (CG) neurons were blocked reversibly by arachidonic acid and a non-metabolizable analog of arachidonic acid, 5,8,11,14-eicosatetraynoic acid. Inhibition of IA by both lipids was observed in whole-cell recordings and in excised inside-out patches, suggesting that Kv4 (Shal) subunits contribute to functional IA channels in CG neurons. Consistent with this, Kv4.2 and Kv4.3 cDNAs were isolated by RT-PCR from chick CG neurons.

Posttranslational regulation of Ca(2+)-activated K+ currents by a target-derived factor in developing parasympathetic neurons.

Macroscopic IK[Ca is not expressed in normal levels in chick ciliary ganglion (CG) neurons prior to synapse formation with target tissues, or in neurons developing in vitro or in situ in the absence of target tissues. Here, two chick CG slo partial cDNAs encoding IK[Ca channels were isolated, cloned, and sequenced. Both slo transcripts were readily detected in developing CG neurons prior to or in the absence of target tissue interactions. When CG neurons developed in vitro in the presence of target tissue (iris) extracts, a normal whole-cell IK[Ca was expressed. These effects did not require protein synthesis, and the activity was detectable throughout the stages of synapse formation in the iris. The active component has an apparent molecular weight of 40-60 kDa.

Information processing in mammalian olfactory system.

In recent years, considerable progress has been made in understanding how the olfactory system uses neural space to encode sensory information. In this review, we focus on recent studies aimed at understanding the organizational strategies used by the mammalian olfactory system to encode information. The odorant receptor gene family is discussed in the context of its genomic organization as well as the specificity of olfactory sensory neurons. These data have important consequences for the mechanisms of odorant receptor gene choice by a given sensory neuron. Division of the olfactory epithelium into zones that express different sets of odorant receptors is the first level of input organization. The topographical relationship between periphery and olfactory bulb represents a further level of processing of information and results in the formation of a highly organized spatial map of information in the olfactory bulb. There, local circuitry refines the sensory input through various lateral interactions. Finally, the factors that may drive the development of such a spatial map are discussed. The onset of expression and the establishment of the zonal organization of odorant receptor genes in the epithelium are not dependent upon the presence of the olfactory bulb, suggesting that the functional identity of olfactory sensory neurons is determined independently of target selection.

Synaptology of the olfactory bulb of an elasmobranch fish, Sphyrna tiburo.

The ultrastructure of the elasmobranch olfactory bulb was examined in order to determine the synaptology of the olfactory circuitry in the bonnethead shark, Sphyrna tiburo. The compartmentalization of the bulb, together with the lack of mitral cell basal dendrites, suggests a different way of performing lateral communication between mitral cells of the olfactory bulb. The results show that granule cells assume an important role by directly interlinking mitral cells. A corollary of this is the segregation of the input onto the mitral cell dendritic arborization: afferent fibers synapse onto the intraglomerular mitral terminals, whereas most local circuit interactions utilize extraglomerular synapses located on the shafts and the somas of the mitral dendrites. Therefore, the elasmobranch synaptic pattern is different from that of higher vertebrates; This might represent the use of a different neural route to achieve the same processing task.

Projections of the olfactory bulb in an elasmobranch fish, Sphyrna tiburo: segregation of inputs in the telencephalon.

We have previously shown that the morphological compartmentalization of the elasmobranch olfactory bulb is accompanied by a topographical arrangement of the primary olfactory projections onto the bulb. If this spatial arrangement is significant for the processing of the information, one would expect it to be preserved in the secondary olfactory centers of the telencephalon. In this paper, we describe the elasmobranch secondary projections from the olfactory bulb to the telencephalon, focusing on their spatial arrangements within the forebrain. Results show that the olfactory input onto the telencephalon are segregated. The medial olfactory tract projects rostrally onto the superficial layer of the dorsal pallium and onto the lateral pallium. The lateral olfactory tract projects caudally onto the lateral pallium, the striatum and the area superficialis basalis. Thus, the secondary olfactory projections are segregated within the telencephalon, with an overlapping of the secondary fibers in the main projection area, the lateral pallium.

Mitral cell dendrites: a comparative approach.

Phylogenetically persistent structures such as the mitral cells of the vertebrate olfactory bulb undergo changes in their dendritic arbor in the course of evolution. The morphology of mitral cells and the main elements of the olfactory bulb circuit in all classes of vertebrates are reviewed in this paper. Most of the neuronal elements found in the mammalian olfactory bulb are present in anamniotes. However, in contrast to those of amniotes, the mitral cells of most anamniotes lack basal dendrites, and periglomerular cells are absent in fish. This suggests a different circuitry and therefore drastic changes in the processing of olfactory information within the olfactory bulb. Lateral inhibition, conferred by basal dendrites in anamniotes, must then utilize other mechanisms in anamniotes. Moreover, the marked segregation of olfactory inputs onto mammalian mitral cells is less obvious in mitral cells of anamniotes that lack basal dendrites. The general role of dendrites, including those of mitral cells, is discussed in the light of increasing evidence for dendritic excitability. The evolutionary significance of mitral cell basal dendrites is also discussed.

Influence of the olfactory organ on brain development.

The olfactory epithelium is a unique sensory structure that has the intrinsic ability to renew its receptor neurons naturally throughout the vertebrate lifetime. The olfactory epithelium is also a neurogenetic matrix that generates various cell populations during embryonic development and adulthood. Some of these cell types migrate to the forebrain and therefore contribute to brain formation. The molecules involved in cell migration and continuous reconnection of olfactory receptor neurons to the bulb are discussed. Experiments involving removal or transplants of the olfactory placode in amphibians suggest that the olfactory organ influences the development of the telencephalon. We believe that the olfactory organ has a morphogenetic influence and even possibly an inducing effect on the forebrain. This effect could be mediated by a number of organizing factors that we discuss. The profound influence of the olfactory organ on brain development underlines the importance of the olfactory organ for survival.

A pilot study on morphological compartmentalization and heterogeneity in the elasmobranch olfactory bulb.

The olfactory bulb of many elasmobranch fishes is morphologically subdivided into distinct units or sub-bulbs immediately adjacent to the olfactory epithelium. We investigated this morphological feature in two species of shark and one species of ray in order to understand its impact on the arrangement of the primary olfactory projections onto the bulb. Using anterograde tracing methods in vitro (biocytin) as well as in fixed tissue (DiI), we observed a direct segregated projection of the olfactory afferents onto the bulb. Application of tracers to the lateral part of the olfactory epithelium resulted in staining restricted to this region of the bulb, whereas the same tracers applied to the medial part of the epithelium resulted in staining of the medial olfactory bulb. The sub-bulbs appear to be individual anatomical units that each receive input from the olfactory lamellae. Nissl and myelin staining as well as the Golgi method show that the cytoarchitecture of the sub-bulbs is not substantially different from that of other anamniotes. However, we did note the existence of two types of mitral cell, based on the morphology of their dendritic arborization. Type L cells exhibit a loose dendritic arborization, whereas type T cells are characterized by a dense, bush-like dendritic arborization. Both types of mitral cells lack basal dendrites.