Developmental regulation of metabotropic glutamate receptor 1 splice variants in olfactory bulb mitral cells.

Alternative splicing of the metabotropic glutamate receptor 1 (mGluR1) receptor gene generates two major receptor isoforms, mGluR1a and mGluR1b, differing in intracellular function and distribution. However, little is known on the expression profiles of these variants during development. We examined the mRNA expression profile of mGluR1a/b in microdissected layers and acutely isolated mitral cells in the developing mouse olfactory bulb. This analysis showed that the two mGluR1 variants are differentially regulated within each bulb layer. During the first postnatal week, the mGluR1a isoform replaces GluR1b in the microdissected mitral cell layer (MCL) and in isolated identified mitral cells, coinciding with a developmental epoch of mitral cell dendritic reorganization. Although mGluR1a mRNA is expressed at high levels in both the adult external plexiform layer (EPL) and MCL, Western blotting analysis reveals a marked reduction of the mGluR1a protein in the MCL, where mitral cell bodies are located, and strong labeling in the EPL, which contains mitral cell dendrites. This suggests that there is increased dendritic trafficking efficiency of the receptor in adult. The temporal and spatial shift in mGluR1b/a expression suggests distinct roles of the mGluR1 isoforms, with mGluR1b potentially involved in the early mitral cell maturation and mGluR1a in dendritic and synapse function.

Expression of the secreted factors noggin and bone morphogenetic proteins in the subependymal layer and olfactory bulb of the adult mouse brain.

The antagonism between noggin and the bone morphogenetic proteins (BMPs) plays a key role during CNS morphogenesis and differentiation. Recent studies indicate that these secreted factors are also widely expressed in the postnatal and adult mammalian brain in areas characterized by different types of neural plasticity. In particular, significant levels of noggin and BMP expression have been described in the rodent olfactory system. In the mammalian forebrain, the olfactory bulb (OB) and associated subependymal layer (SEL) are documented as sites of adult neurogenesis. Here, using multiple approaches, including the analysis of noggin-LacZ heterozygous mice, we report the expression of noggin and two members of the BMP family, BMP4 and BMP7, in these regions of the adult mammalian forebrain. We observe that along the full extent of the SEL, from the lateral ventricle to the olfactory bulb, noggin and BMP4 and 7 are mainly associated with the astrocytic glial compartment. In the OB, BMP4 and 7 proteins remain primarily associated with the SEL while strong noggin expression was also found in cells located in different OB layers (i.e. granule, external plexiform, glomerular layers). Taken together our data lead us to hypothesize that within the SEL the antagonism between noggin and BMPs, both produced by the glial tubes, act through autocrine/paracrine inductive mechanisms to maintain a neurogenetic environment all the way from the lateral ventricle to the olfactory bulb. In the OB, their expression patterns suggest multiple regulatory roles on the unusual neural plasticity exhibited by this region.

OMP gene deletion results in an alteration in odorant-induced mucosal activity patterns.

Previous behavioral work, using a complex five-odorant identification task, demonstrated that olfactory marker protein (OMP) is critically involved in odor processing to the extent that its loss results in an alteration in odorant quality perception. Exactly how the lack of OMP exerts its influence on the perception of odorant quality is unknown. However, there is considerable neurophysiological evidence that different odorants produce different spatiotemporal patterns of neural activity at the level of the mucosa and that these patterns predict the psychophysically determined perceptual relationship among odorants. In this respect, OMP gene deletion is known to result in a constellation of physiologic defects (i.e., marked reduction in the electroolfactogram (EOG) and altered response and recovery kinetics) that would be expected to alter the odorant-induced spatiotemporal activity patterns that are characteristic of different odorants. This, in turn, would be expected to alter the spatiotemporal patterning of information that results from the mucosal projection onto the bulb, thereby changing odorant quality perception. To test the hypothesis that odorant-induced mucosal activity patterns are altered in mice lacking the gene for OMP, we optically recorded the fluorescent changes in response to odorant stimulation from both the septum and turbinates of both OMP-null and control mice using a voltage-sensitive dye (di-4-ANEPPS Molecular Probes, Eugene, OR) and a Dalsa 120 x 120, 12-bit CCD camera. To maintain continuity with the previous behavioral work, the odorants 2-propanol, citral, carvone, ethylacetoacetate, and propyl acetate were again used. Each odorant was randomly presented to each mucosal surface in a Latin-Square design. The results of this study demonstrated that, for both mouse strains, there do indeed exist different spatiotemporal activity patterns for different odorants. More importantly, however, these patterns significantly differed between OMP-null and control mice. That is, although the general regions of characteristic activity for different odorants were the same in both mouse strains, the patterns in the null animals were degraded relative to controls. These data suggest therefore that the alterations in mucosal activity may serve as the substrate for the behaviorally observed changes in odorant quality perception in the null mutant.

Dopamine D2 receptor-mediated presynaptic inhibition of olfactory nerve terminals.

Olfactory receptor neurons of the nasal epithelium project via the olfactory nerve (ON) to the glomeruli of the main olfactory bulb, where they form glutamatergic synapses with the apical dendrites of mitral and tufted cells, the output cells of the olfactory bulb, and with juxtaglomerular interneurons. The glomerular layer contains one of the largest population of dopamine (DA) neurons in the brain, and DA in the olfactory bulb is found exclusively in juxtaglomerular neurons. D2 receptors, the predominant DA receptor subtype in the olfactory bulb, are found in the ON and glomerular layers, and are present on ON terminals. In the present study, field potential and single-unit recordings, as well as whole cell patch-clamp techniques, were used to investigate the role of DA and D2 receptors in glomerular synaptic processing in rat and mouse olfactory bulb slices. DA and D2 receptor agonists reduced ON-evoked synaptic responses in mitral/tufted and juxtaglomerular cells. Spontaneous and ON-evoked spiking of mitral cells was also reduced by DA and D2 agonists, and enhanced by D2 antagonists. DA did not produce measurable postsynaptic changes in juxtaglomerular cells, nor did it alter their responses to mitral/tufted cell inputs. DA also reduced 1) paired-pulse depression of ON-evoked synaptic responses in mitral/tufted and juxtaglomerular cells and 2) the amplitude and frequency of spontaneous, but not miniature, excitatory postsynaptic currents in juxtaglomerular cells. Taken together, these findings are consistent with the hypothesis that activation of D2 receptors presynaptically inhibits ON terminals. DA and D2 agonists had no effect in D2 receptor knockout mice, suggesting that D2 receptors are the only type of DA receptors that affect signal transmission from the ON to the rodent olfactory bulb.

OMP gene deletion results in an alteration in odorant quality perception.

To test the hypothesis that odorant quality perception is altered in olfactory marker protein (OMP)-null mice, we trained and tested adult OMP-null and control mice, using a 5-odorant identification confusion matrix task (animal odorant confusion matrix [AOCM]). On average, control and null mice performed the task at equivalent levels. The composite 5 x 5 response matrix from 40 testing sessions for each subject (both OMP-null and control) was compared with that of every other subject, yielding a dissimilarity matrix of AOCM responses. A multidimensional scaling (MDS) analysis of the dissimilarity data yielded a 4-dimensional solution, with each mouse occupying a point in MDS animal space. Statistical analysis demonstrated significant effects of genotype in determining the location of a mouse in the MDS space. These data suggest, therefore, that compared with that of controls, odorant quality perception is altered in the OMP-null mouse.

The OMP-lacZ transgene mimics the unusual expression pattern of OR-Z6, a new odorant receptor gene on mouse chromosome 6: implication for locus-dependent gene expression.

Reporter gene expression in the olfactory epithelium of H-lacZ6 transgenic mice mimics the cell-selective expression pattern known for some odorant receptor genes. The transgene construct in these mice consists of the lacZ coding region, driven by the proximal olfactory marker protein (OMP) gene promoter, and shows expression in a zonally confined subpopulation of olfactory neurons. To address mechanisms underlying the odorant receptor-like expression pattern of the lacZ construct, we analyzed the transgene-flanking region and identified OR-Z6, the first cloned odorant receptor gene that maps to mouse chromosome 6. OR-Z6 bears the highest sequence similarity (85%) to a human odorant receptor gene at the syntenic location on human chromosome 7. We analyzed the expression pattern of OR-Z6 in olfactory tissues of H-lacZ6 mice and show that it bears strong similarities to that mapped for beta-galactosidase. Expression of both genes in olfactory neurons is primarily restricted to the same medial subregion of the olfactory epithelium. Axons from both neuronal subpopulations project to the same ventromedial aspect of the anterior olfactory bulbs. Furthermore, colocalization analyses in H-lacZ6 mice demonstrate that OR-Z6-reactive glomeruli receive axonal input from lacZ-positive neurons as well. These results suggest that the expression of both genes is coordinated and that transgene expression in H-lacZ6 mice is regulated by locus-dependent mechanisms.

The esthetic space maintainer.

This article describes why it is important to replace the maxillary primary incisors when they are prematurely lost in children. The consequences of not replacing these teeth in relation to speech, esthetics, and self-image considerations are provided. A step-by-step description is presented for the fabrication of an esthetic appliance, which can be easily made to replace missing maxillary primary incisors. In addition, information is presented on when and how to remove the esthetic appliance.

Adenoviral vector-mediated rescue of the OMP-null phenotype in vivo.

The use of gene deletion by homologous recombination to determine gene or protein function has wide application in vertebrate neurobiology. An ideal complement to gene deletion would be subsequent gene replacement to demonstrate re-acquisition of function. Here we used an adenoviral vector to replace the olfactory marker protein (OMP) gene in olfactory receptor neurons of adult OMP-null mice and demonstrated the subsequent re-acquisition of function. Our results show that short-term expression of OMP restores the kinetics of electrophysiological responses of OMP-null mice to those of the control phenotype. This adenoviral-mediated rescue of the OMP-null phenotype is consistent with involvement of OMP in olfactory transduction.

Carnosine-related dipeptides in neurons and glia.

Carnosine-related dipeptides have been demonstrated to occur in the nervous tissue of many vertebrates, including humans. Although several hypotheses have been formulated, to date their precise physiological role in the nervous system remains unknown. This article will review the studies on the presence and distribution of these dipeptides in the nervous system of different classes of vertebrates. It will focus on the most recent data on their cellular localization and potential functions in mammals. The studies on localization of carnosine-related dipeptides show a complex pattern of expression that involves both neuronal and glial cell types. The glial localization, widely distributed throughout the whole brain and spinal cord, includes a subset of both mature astrocytes and oligodendrocytes, whereas the neuronal localization is restricted to a particular type of neurons (the olfactory receptor neurons), and to restricted populations of putative migrating neurons and neuroblasts. There is no definitive demonstration of the function of these dipeptides in the various cell types. However, a wide array of evidence suggests that carnosine-related dipeptides could act as natural protective agents. Moreover, recent studies have suggested that, as previously postulated for the olfactory receptor neurons, in mature functional glial cells as well, carnosine-related dipeptides could be implicated in a neuromodulatory functional mechanism.

Ultrastructural characteristics and conduction velocity of olfactory receptor neuron axons in the olfactory marker protein-null mouse.

Olfactory receptor neuron (ORN) axon diameters and the conduction velocity of the compound action potential along ORN axons were studied in olfactory marker protein (OMP)-null mice and genotypically matched controls. The compound action potential was distinguished from postsynaptic field potentials by its shorter latency, its persistence following application of cobalt or kynurenic acid that blocked postsynaptic responses, and its ability to follow paired-pulse stimulation at 300 Hz. Blockade of the postsynaptic field responses by kynurenic acid indicates that in the mouse, as in the rat, glutamate is the olfactory nerve transmitter. The mean conduction velocity of ORNs in wild-type control mice was 0. 47+/-0.19 (S.E.M.) m/s (n=5), similar to the conduction velocity reported for other mammals. The mean diameter of ORN axons in control mice was 0.202+/-0.005 and 0.261+/-0.006 microm in the OMP-null mice. This increase in fiber diameter in the OMP-nulls predicts an increase in impulse conduction velocity. However, the mean conduction velocity of OMP-null mice, 0.38+/-0.03 m/s (n=6), was not significantly different from control (P>0.1). The conduction velocity predicted by the increase in fiber diameter in OMP-null mice was within the 95% confidence interval of the measured value. Thus, OMP-null ORNs are normal with respect to the conduction velocity of their axons. The number of axodendritic synapses in the glomeruli of OMP-null mice is higher than in congenic wild-type mice.

Immunohistochemical distribution of galectin-1, galectin-3, and olfactory marker protein in human olfactory epithelium.

The expression pattern of galectin-1 and galectin-3 in the human olfactory epithelium was investigated in relation to olfactory marker protein (OMP) using confocal laser immunofluorescence in human specimens and postmortem biopsies. OMP expression was found in olfactory receptor neurons (ORNs) in the olfactory mucosa and in fibers of the olfactory nerve crossing the submucous connective tissue. Galectin-1 was expressed in both the connective tissue of the nasal cavity and in the basal layer of the olfactory epithelium. In contrast, galectin-3 expression was limited to cells of the upper one-third of the olfactory epithelium. Expression of galectin-3 occurred in a subset of OMP-positive cells. However, between areas of galectin-1 and galectin-3 expression in the lower and upper portion of the epithelium, OMP-positive ORNs did not stain for both galectins. Considering the potential role of galectin-1 and galectin-3 in cell differentiation and maturation, the differential localization of galectins in the olfactory epithelium appears to be consistent with a significant role of these molecules in the physiological turnover of ORNs.

Pattern of olfactory bulb innervation returns after recovery from reversible peripheral deafferentation.

The olfactory epithelium (OE) is unusual in its ability to regenerate and reinnervate its target, the olfactory bulb (OB), after deafferentation. To address the question of whether olfactory receptor neuron (ORN) axons preserve their topographic organization when they reestablish synaptic contact with the OB, the authors examined the pattern of ORN axon reinnervation into the bulb of adult H-OMP-lacZ-6 transgenic mice during and after recovery from chemical deafferentation. In the H-OMP-lacZ-6 mouse strain, lacZ expression is limited to a subset of ORNs that are distributed bilaterally in the OE and project primarily to a few glomeruli in the ventromedial region of the OB. The OE was lesioned by intranasal irrigation with Triton X-100, and the distribution of 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal)-stained cells was examined in the OE along with beta-galactosidase-immunoreactive (beta-gal-ir) axonal processes in the OB after short (1 week), intermediate (3 week), and long (6-7 weeks) recovery times. One week after the lesion, immunostaining for beta-gal and olfactory marker protein was virtually eliminated in the bulb. After 3 weeks of recovery, beta-gal-containing axons appeared to target many of the same locations innervated in bulbs of unlesioned mice. The region that received the highest density of axonal innervation in controls, however, contained only a few processes at that time. After 6-7 week recovery periods, the pattern of X-gal staining in the OE and beta-gal-ir axons in the OB closely resembled that of unlesioned mice. These results demonstrate that the topographic distribution of ORNs in the OE and the pattern of axon innervation in the OB can be reconstituted after chemical deafferentation.

NFI in the development of the olfactory neuroepithelium and the regulation of olfactory marker protein gene expression.

Nuclear factor I (NFI) proteins are DNA-binding transcription factors that participate in the tissue specific expression of various genes. They are encoded by four different genes (NFI-A, B, C, and X) each of which generates multiple isoforms by alternative RNA splicing. NFI-like binding sites have been identified in several genes preferentially expressed in olfactory receptor neurons. Our prior demonstration that NFI binds to these elements led to the hypothesis that NFI is involved in the regulation of these genes. To analyse the role of NFI in the regulation of olfactory neuron gene expression we have performed transient transfection experiments in HEK 293 cells using constructs that place luciferase expression under the control of an olfactory marker protein (OMP)-promoter fragment containing the NFI binding site. In vitro mutagenesis of this site revealed a negative modulation of luciferase expression by endogenous NFI proteins in HEK 293 cells. In addition, we have used in situ hybridization to analyse the tissue and cellular distribution of the four NFI gene transcripts during pre- and postnatal mouse development. We have simultaneously characterized the expression of Pax-6, and O/E-1, transcription factors known to regulate the phenotype of olfactory receptor neurons. We demonstrate that all of these transcription factors vary in specific spatio-temporal patterns during the development of the olfactory system. These data on NFI activity, and on transcription factor expression, provide a basis to understand the role of NFI in regulating gene expression in olfactory receptor neurons.

Identification of NFI-binding sites and cloning of NFI-cDNAs suggest a regulatory role for NFI transcription factors in olfactory neuron gene expression.

Olfactory receptor neurons are responsible for the detection and signal transduction of odor ligands. Several genes associated with this activity are preferentially or exclusively expressed in these neurons. Among these genes are those coding for olfactory receptors, adenylyl cyclase type III, the cyclic nucleotide gated olfactory channel 1 (OcNC-1), Galpha(olf) and the olfactory marker protein (OMP). Promoter analyses of these genes identified a binding site for the new transcription factor family O/E whose initial member, Olf-1, is abundantly expressed in olfactory neurons. We report here that the proximal promoters of three of these genes, that are selectively expressed in olfactory neurons, each contains a functional NFI binding site and that the sites have different affinities for NFI proteins indicating a regulatory role for NFI proteins in olfactory gene expression. We further demonstrate, by cloning, that all four NFI genes are expressed in the olfactory nasal mucosa. Analysis by in situ hybridization illustrates that at least three of these gene products are expressed in the neuroepithelium in which the olfactory neurons reside. NFI proteins are capable of functioning as positive or negative regulators of transcription depending on the tissue, cell-type, age, and gene in question. These multivalent functions of NFI could be achieved by temporally and spatially regulated expression of distinct subsets of NFI isoforms. It now remains to characterize the tissue and cell specific patterns of expression of distinct NFI transcription factors during ontogeny and their roles in regulating gene expression.

Targeted deletion of a cyclic nucleotide-gated channel subunit (OCNC1): biochemical and morphological consequences in adult mice.

The olfactory cyclic nucleotide-gated channel subunit 1 (OCNC1) is required for signal transduction in olfactory receptor cells. To further investigate the role of this channel in the olfactory system, the biochemical and morphological consequences of targeted disruption of OCNC1 were investigated in adult mice. Null as compared to wild-type mice had smaller olfactory bulbs, suggesting compromised development of the central target of the receptor cells. Ectopic olfactory marker protein (OMP)-stained fibers localized to the external plexiform layer reflected the relative immaturity of the olfactory bulb in the null mice. The olfactory epithelium of the knock-out mouse was thinner and showed lower expression of olfactory marker protein and growth-associated protein 43, indicating decreases in both generation and maturation of receptor cells. Tyrosine hydroxylase (TH) expression in the olfactory bulb, examined as a reflection of afferent activity, was reduced in the majority of periglomerular neurons but retained in atypical or "necklace" glomeruli localized to posterior aspects of the olfactory bulb. Double label studies demonstrated that the remaining TH-immunostained neurons received their innervation from a subset of receptor cells previously shown to express a phosphodiesterase that differs from that found in most receptor cells. These data indicate that expression of OCNC1 is required for normal development of the olfactory epithelium and olfactory bulb. The robust expression of TH in some periglomerular cells in the OCNC1-null mice suggests that receptor cells innervating these glomeruli may use an alternate signal transduction pathway.

Taste processing: whetting our appetites.

Two G-protein-coupled receptors have been identified that are present in the apical membranes of rat and mouse taste cells and differentially distributed across the tongue and palate. They are strong candidates for being taste receptors and their discovery has provided new tools for research into gustatory processing.