Glutamatergic deafferentation of olfactory bulb modulates the expression of mGluR1a mRNA

Glutamate (Glu) released by olfactory nerve axons acts on postsynaptic ionotropic and metabotropic glutamate receptors expressed by principal neurones and interneurones of the olfactory bulb (OB). Using ZnSO4 lesioning of the rat olfactory mucosa and semiquantitative RT-PCR, we examined the effect of removal of the glutamatergic input to the OB on the expression of mGluR1a, mGluR1b and GluR1 mRNAs. Two days after lesioning, mGluR1a mRNA levels in OB increased by 45%. At this time, the expression of tyrosine hydroxylase (TH) mRNA, which is strictly dependent on olfactory nerve input, was still unchanged. In contrast, 16 days after lesioning, deafferented OB exhibited a decrease in both mGluR1a (-30%) and TH (-40%) mRNAs. GluR1 and mGluR1b mRNA levels were not affected at either time point. These results suggest that alterations in glutamatergic input to OB selectively modulate the expression of the mGluR1 splicing form possessing a longer C-terminal domain.     

Cell-specific differential expression of Na(+)-channel beta 1-subunit mRNA in the olfactory system during postnatal development and after denervation

Activity-dependent mechanisms have been implicated in olfactory system development but, although such activity requires ion channels, few reports have described their expression in the olfactory system. We investigated the developmental and denervation-induced regulation of the Na(+)-channel beta 1 subunit (Na beta 1) in rat olfactory bulb (OB) and piriform cortex (PC). In situ hybridization shows that Na beta 1 mRNA expression is upregulated developmentally, but with different time courses in mitral, tufted, and pyramidal cells. In mitral cells, label was detected at postnatal day 4 (P4) and gradually increased to P14. Tufted cells were devoid of Na beta 1 mRNA before P14, when most cells expressed adult levels. In pyramidal cells of PC, Na beta 1 expression was not detectable clearly until P14, with maximal expression at P28. To examine the regulation of Na beta 1 mRNA, we surgically deafferented the OB at P30 and compared the effects on Na beta 1 with those for Na(+)-channel alpha-subunit (Na alpha) mRNAs. Within 5 d of surgery, the Na beta 1 and Na alpha II signals within tufted cells disappeared almost completely. Na beta 1 and Na alpha II expression was decreased in mitral cells to low-to-moderate levels. In pyramidal cells, Na beta 1 mRNA expression was decreased moderately without significant changes in Na alpha II mRNA. Deafferentation had no detectable effects on Na alpha I or III mRNAs in either OB or PC. These data indicate that Na beta 1 mRNA is expressed differentially in subpopulations of cells in the olfactory system during development and after deafferentation and suggest that the expression of Na beta 1 is regulated independently of Na alpha mRNAs via cell-specific and pathway-specific mechanisms.     

Bidirectional processing in the olfactory-limbic axis during olfactory behavior

Field potentials were recorded simultaneously from the olfactory bulb (OB), prepyriform cortex (PPC), entorhinal cortex (EC), and dentate gyrus (DG) of rats trained to respond to appetitively reinforced odors. Preafferent anticipatory events in the beta band (12-35 Hz) suggest transmission from EC to OB before the odorant stimulus. Gamma band (35-120 Hz) power in olfactory regions is significantly reduced during stimulus presentation as compared with high values during preafferent expectation. High coherence of OB and PPC gamma activity during the preodorant control period is interrupted before the stimulus and is followed by increased gamma coherence among OB, EC, and DG. These results suggest that olfactory perceptual processing is bidirectional and covers a wide frequency range.     

The expression of B-50/GAP-43 and GFAP after bilateral olfactory bulbectomy in rats

In the present study we investigated the effect of a two-stage bilateral lesion of the olfactory bulb (OB) in rats on the regeneration ability of peripheral olfactory neurons and their reinnervation capacity in the spared OB. The outgrowth of newly-generated olfactory axons as well as the maturation of their terminal synaptic field was detected by immunohistochemistry of the growth-associated phosphoprotein B-50/GAP-43. In addition, the glial response to the surgery was monitored by an immunohistochemical marker for astrocytes, glial fibrillary acidic protein (GFAP). In neonatal rats (P3-P5), the right OB was removed, then three months later the contralateral side was ablated. Six days after the second operation the animals were transcardially perfused. Their brains were embedded in paraplast, serially sectioned and processed for histological and immunohistochemical observations. After neonatal OB ablation, homogeneous B-50-immunoreactivity (BIR) was found in the forebrain, olfactory axons and ectopic glomeruli localized in the small OB remnant-like structures and in the regenerated neuroepithelium. A strong GFAP response was revealed in the brain cortex as well as in the newly-formed olfactory axons and glomeruli-like structures of the OB remnants. After adult OB ablation strong BIR was observed in olfactory axons, while remaining glomerular structures were only faintly stained. The neuroepithelium revealed signs of massive degenerative processes with a substantial decrease in BIR. The GFAP-positive astrocytes were scattered throughout the entire OB remnant and were prominent in the glomeruli-like structures and adjacent frontal cortex. In the present study, we applied GAP-43 and GFAP immunohistochemistry to characterize the responses of individual olfactory components after two-stage olfactory bulbectomy. Furthermore, this model of OB ablation characterized by two immunohistochemical markers could elucidate certain molecular mechanisms involved in the regeneration and/or plasticity of the olfactory system.     

Odorant response properties of convergent olfactory receptor neurons

Information about odorant stimuli is thought to be represented in spatial and temporal patterns of activity across neurons in the olfactory epithelium and the olfactory bulb (OB). Previous studies suggest that olfactory receptor neurons (ORNs) distributed in the nasal cavity project to localized regions in the glomerular layer of the OB. However, the functional significance of this convergence is not yet known, and in no studies have the odorant response properties of individual ORNs projecting to defined OB regions been measured directly. We have retrogradely labeled mouse ORNs connecting to different glomeruli in the dorsal OB and tested single cells for responses to odorants using fura-2 calcium imaging. ORNs that project to clusters of dorsomedial (DM) glomeruli exhibit different odorant response profiles from those that project to dorsolateral (DL) glomeruli. DL-projecting ORNs showed responses to compounds with widely different structures, including carvone, eugenol, cinnamaldehyde, and acetophenone. In contrast, DM-projecting neurons exhibited responses to a more structurally restricted set of compounds and responded preferentially to organic acids. These data demonstrate that ORN afferents segregate by odorant responsiveness and that the homogeneity of ORN and glomerular input varies with different OB regions. The data also demonstrate that a subpopulation of ORNs projecting to DM glomeruli is functionally similar.     

Importance of !b-noradrenergic receptors in the olfactory bulb of sheep for recognition of lambs.

Within a 4-hr period after parturition, the ewe learns the odor of her lamb that will allow later recognition of her offspring from alien lamb. This study investigated the importance of the activation of olfactory bulb (OB) β-noradrenergic receptors in establishing this olfactory memory. Pregnant ewes (n?=?24) were cannulated bilaterally in the OB. Two days before parturition, OB were perfused continuously (10 μl/hr) with either a solution containing the selective β-noradrenergic antagonist propranolol (10–3 M) or vehicle. The results showed that, although 8 of 9 control ewes were still able to bond selectively to their lambs, only 7 of 15 propranolol-treated animals were able to bond (p?=?.04). These results suggest that activation of β-noradrenergic receptors in the OB during the period surrounding parturition is required to establish olfactory recognition of the lamb. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Establishment and neurite outgrowth properties of neonatal and adult rat olfactory bulb glial cell lines

Two glial cell types surround olfactory axons and glomeruli in the olfactory bulb (OB) and may influence synapse development and regeneration. OB astrocytes resemble type-1 astrocytes, and OB ensheathing cells resemble non-myelinating Schwann cells. We have produced clonal OB astrocyte and ensheathing cell lines from rat neonatal and adult OB cultures by SV40 large T antigen transduction. These cell lines have been characterized by morphology, growth characteristics, immunophenotype, and ability to promote neurite outgrowth in vitro. Neonatal and adult ensheathing cell lines were found to support higher neurite outgrowth than OB astrocyte lines. Neonatal OB astrocyte lines were of two types, high and low outgrowth support. The low support astrocyte lines express J1 and a chondroitin sulfate-containing proteoglycan as do astrocytes encircling the neonatal glomeruli in vivo. The adult OB astrocyte cell lines supported lower levels of outgrowth than adult ensheathing cell lines. These results are consistent with a positive role for ensheathing cells in OB synapse regeneration, in vivo. Further, based on our results, we hypothesize that ensheathing cells and high-outgrowth astrocytes facilitate axon growth in vivo, while low outgrowth astrocytes inhibit axon growth and may facilitate glomerulus formation.     

Noradrenergic neuromodulation in the olfactory bulb modulates odor habituation and spontaneous discrimination.

Noradrenergic projections from the locus coeruleus (LC) project to the olfactory bulb (OB), a cortical structure implicated in odor learning and perceptual differentiation among similar odorants. The authors tested the role of OB noradrenaline (NA) in short-term olfactory memory using an animal model of LC degeneration coupled with intrabulbar infusions of NA. Specifically, the authors lesioned cortical noradrenergic fibers in mice with the noradrenergic neurotoxin N-Ethyl-N-(2-chloroethyl)-2-bromobenzylamine hydrochloride (DSP4) and measured the effects on an olfactory habituation/spontaneous discrimination task. DSP4-treated mice failed to habituate to repeated odor presentations, indicating that they could not remember odors over the 5-min intertrial interval. The authors then infused NA bilaterally into the OBs of both DSP4-treated and nonlesioned control animals at two concentrations (10-3M and 10-5M, 2 μl/side). In DSP4-treated animals, NA administration at either concentration restored normal habituation and spontaneous discrimination performance, indicating that noradrenergic neuromodulation mediates these aspects of perceptual learning and that its efficacy does not require activity-dependent local regulation of NA release. Functional OB learning mechanisms may be necessary for normal odor recognition and differentiation among physically similar odorants. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Two novel monoclonal antibodies (1.9.E and 4.11.C) against olfactory bulb ensheathing glia

We produced and characterized two monoclonal antibodies, termed 1.9.E and 4.11.C, that specifically recognize olfactory bulb ensheathing glia. Both antibodies were generated using the olfactory nerve layer (ONL) of newborn rat olfactory bulbs (P0, P1) as immunogens. The specificity of these antibodies was tested by immunofluorescence techniques on tissue sections and cultures of adult and neonatal rat olfactory bulbs, and by Western blot analysis. 1.9.E labeled the ONL and glomerular layer of the olfactory bulb (OB) of adult rats. In newborn rats, 1.9.E immunostained ensheathing cells from the ONL and peripheral olfactory fascicles. Furthermore, 1.9.E reacted with some processes of the radial glia in the periventricular germinal layer of the newborn rat. Although 4.11.C also specifically labeled ensheathing cells in the adult OB, it did not stain any cell type in the ONL of newborn rats. The lack of double labeling with either 1.9.E or 4.11.C and anti-olfactory marker protein (OMP) antibody, a specific marker for olfactory axons, indicated that none of the monoclonals recognized olfactory axons. Double immunostaining of adult OB cultures with 1.9.E or 4.11.C and anti-p75-nerve growth factor receptor revealed that both antibodies specifically recognized ensheathing glia in those cultures. Filaments were strongly labeled throughout the entire cytoplasm of ensheathing cells, suggesting that 1.9.E and 4.11.C immunoreacted with ensheathing glia cytoskeleton. 4.11.C stained a few Schwann cells in adult sciatic nerve sections. Moreover, 4.11.C immunostained cortical astrocyte cultures from newborn rats (P1). In Western blot analysis both antibodies recognized a major component, migrating with an apparent molecular weight of 60 kDa, from olfactory nerve and glomerular layer (ONGL) extracts of adult and neonatal rats. The pattern of immunoreactivity of 1.9.E and 4.11.C antibodies suggest that both antibodies are specific markers for olfactory ensheathing glia in the adult rat central nervous system (CNS).     

Sodium current in periglomerular cells of rat olfactory bulb in vitro

The capacity of periglomerular cells (PGc) to give fast, Na-dependent action potentials is a crucial and debated issue for the comprehension of how sensory information is processed in the olfactory bulb (OB). Using patchclamp whole cell recording in thin slices of rat OB (P8-P20) we showed that fast sodium conductance is present in all the PGc studied, that this current is sufficiently large to generate action potentials and that action potentials can be evoked in these cells by direct stimulation of the olfactory nerve. A comprehensive kinetic characterization of INa is also presented.     

Neurocalcin immunoreactivity in the rat main olfactory bulb

The morphological characteristics and distribution of neurocalcin (NC)-immunoreactive elements were studied in the rat main olfactory bulb (OB) using a polyclonal antibody and the avidin-biotin immunoperoxidase method. NC-positive elements were abundant in the glomerular layer (GL), where numerous immunostained external tufted cells and periglomerular cells were detected. Other less abundant NC-immunolabeled populations included middle and internal tufted cells, Van Gehuchten cells, horizontal cells, vertical cells of Cajal, deep short-axon cells and granule cells. This study demonstrates the presence of NC immunoreactivity in subsets of different neuronal types in the rat main OB. This calcium-binding protein has been found in interneurons, and no evidence of immunoreactivity to NC is detected in projecting neurons. Despite the large population of labeled external tufted cells, most of them belong according to morphological criteria to the local circuit group and some others to those with interbulbar and/or intrabulbar connections. The identification of neuronal subpopulations expressing NC provides a further characterization and shows the existence of biochemical differences within morphologically identical neurons. Thus, this marker may be a useful tool in unravelling the circuitries of the rodent OB in both normal and experimental conditions. The exact physiological function of NC in the olfactory system remains unknown. On the basis of similarities to recoverin, it could be involved in mechanisms responsible for sensory adaptation. Additionally, its calcium-binding abilities may contribute to improve the temporal precision of stimuli transmission, or be concerned with general calcium-related events occurring in specific interneuronal groups.     

Chemotopic, combinatorial, and noncombinatorial odorant representations in the olfactory bulb revealed using a voltage-sensitive axon tracer

Odor information is first represented in the brain by patterns of input activity across the glomeruli of the olfactory bulb (OB). To examine how odorants are represented at this stage of olfactory processing, we labeled anterogradely the axons of olfactory receptor neurons with the voltage-sensitive dye Di8-ANEPPQ in zebrafish. The activity induced by diverse natural odorants in afferent axons and across the array of glomeruli was then recorded optically. The results show that certain subregions of the OB are preferentially activated by defined chemical odorant classes. Within these subregions, "ordinary" odorants (amino acids, bile acids, and nucleotides) induce overlapping activity patterns involving multiple glomeruli, indicating that they are represented by combinatorial activity patterns. In contrast, two putative pheromone components (prostaglandin F2alpha and 17alpha, 20beta-dihydroxy-4-pregnene-3-one-20-sulfate) each induce a single focus of activity, at least one of which comes from a single, highly specific and sensitive glomerulus. These results indicate that the OB is organized into functional subregions processing classes of odorants. Furthermore, they suggest that individual odorants can be represented by "combinatorial" or "noncombinatorial" (focal) activity patterns and that the latter may serve to process odorants triggering distinct responses such as that of pheromones.     

Combinatorial and chemotopic odorant coding in the zebrafish olfactory bulb visualized by optical imaging

Odors are thought to be represented by a distributed code across the glomerular modules in the olfactory bulb (OB). Here, we optically imaged presynaptic activity in glomerular modules of the zebrafish OB induced by a class of natural odorants (amino acids [AAs]) after labeling of primary afferents with a calcium-sensitive dye. AAs induce complex combinatorial patterns of active glomerular modules that are unique for different stimuli and concentrations. Quantitative analysis shows that defined molecular features of stimuli are correlated with activity in spatially confined groups of glomerular modules. These results provide direct evidence that identity and concentration of odorants are encoded by glomerular activity patterns and reveal a coarse chemotopic organization of the array of glomerular modules.     

Excitatory synapses in the glomerular triad of frog olfactory bulb in vitro

Whole-cell patch clamp recording techniques were applied to periglomerular (PG) cells in slices of the frog olfactory bulb (OB) to study the properties of the excitatory synapses in the triad formed by the olfactory nerve (ON) and the dendrites of mitral/tufted (MT) cells and PG cells. The postsynaptic response evoked by ON stimulation was glutamatergic and could be dissected into NMDA and non-NMDA components of equivalent amplitudes. The dendro-dendritic synapse between MT and PG cells could be activated following antidromic stimulation of the lateral and medial olfactory tract (LOT and MOT). In this case the postsynaptic potentials had amplitudes and durations comparable to those obtained by ON stimulation, the neurotransmitter was glutamate, but the synapse was largely dominated by the slow NMDA component.     

Functional and molecular characterization of individual olfactory neurons

To gain an understanding of the olfactory signal transduction process, individual chemosensory neurons have been assessed for odor-induced Ca2+ responses and the molecular elements of transduction cascades using Ca2+ imaging technique in combination with single-cell RT-PCR approaches. It has been demonstrated that responsiveness of cells to cyclic AMP or inositol trisphosphate odorants was blocked by specific adenylyl cyclase inhibitors or phospholipase C inhibitors, respectively. Using specific primers in single-cell RT-PCR analysis, olfactory marker protein, two G protein subtypes (G(olf) and G(o)), and adenylyl cyclase (subtype III) and a phospholipase C (phospholipase Cbeta2-related subtype) were identified. For a subpopulation of sensory neurons it was demonstrated that both transduction cascades coexist and are active in the same cell. These data support the notion that two second messenger pathways are active in olfactory sensory neurons and emphasize the concept of dual transduction cascades in olfaction.     

GABAergic neurons in the embryonic olfactory pit/vomeronasal organ: maintenance of functional GABAergic synapses in olfactory explants

In previous work, we showed a robust gamma-aminobutyric acid (GABAergic) synaptic input onto embryonic luteinizing hormone-releasing hormone (LHRH) neurons maintained in olfactory explants. In this study, we identify GABAergic neurons in olfactory pit (OP) of embryonic mice in vivo and study, using patch-pipet whole-cell current and voltage clamp techniques, synaptic interactions of these neurons in explant cultures. In vivo, glutamate decarboxylase (GAD, the enzyme which synthesizes GABA) mRNA was first detected in nasal regions on Embryonic Day (E) 11.5. From E12.5 to E13.5, robust GAD expression was localized to cells primarily in the ventral aspect of the OP. GAD mRNA was not detected over dorsally located cells in olfactory sensory or respiratory epithelium. In addition, GAD mRNA was not observed in cells along olfactory axons. GAD mRNA was dramatically reduced in the OP/vomeronasal organ by E16.5. Using antibodies against both GABA and GAD, immunopositive axonal-like tracts were detected in the nasal septum on E12.5. GABAergic staining decreased by E13.5. To examine synaptic interactions of these GABAergic cells, embryonic olfactory explants were generated and maintained in serum-free media. As explants spread, neuron-like cells migrated into the periphery, sometimes forming ganglion-like clusters. Cells were recorded, marked intracellularly with Lucifer Yellow and post-fixation, immunocytochemically examined. Forty-six cells, typically multipolar, were GABAergic, had resting potentials around -50 mV, and exhibited spontaneous action potentials which were generated by spontaneous depolarizing GABAergic (GABAA) synaptic activity. OP neurons depolarized in response to GABA by increasing Cl- conductance. The biophysical properties of OP-derived GABAergic neurons were distinct from those reported for olfactory receptor neurons but similar to embryonic LHRH neurons. However, unlike LHRH neurons, GABAergic neurons did not migrate large distances in olfactory explants or appear to leave the olfactory pit in vivo.     

Effects of parturition and maternal experience on noradrenaline and acetylcholine release in the olfactory bulb of sheep.

Acetylcholine (ACh) and noradrenaline (NA) release in the olfactory bulb (OB) of ewes was monitored using microdialysis. Both ACh and NA release increased at parturition in multiparous but not in primiparous ewes. However, vaginocervical stimulation performed 6 hrs postpartum induced an increase of ACh and NA release in both primiparous and multiparous ewes, indicating that a maturation process had occurred. Finally, pharmacological challenges to the ACh and NA inputs revealed differential responsiveness between nulliparous and multiparous nongestant ewes. These results suggest that the 1st parturition induces changes in neural circuitry involving ACh and NA inputs to the OB. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Pharmacokinetics and plasma protein binding of sulfamethoxypyridazine in camels (Camelus dromedarius)

Transplant-to-host neuron migration and neurite projection were demonstrated using the mouse allelic Thy-1 system, namely, BALB/c (Thy-1.2) embryonic olfactory bulb (OB) as the graft and 5- to 6-week-old AKR (Thy-1.1) OB as the host. From OB transplants inserted into the host OB, small neurons were often extensively moved mainly in the internal granular layer and showed almost the same morphology as the normal granule neurons. Some large neurons also migrated. Furthermore, inside OB the transplants sent axons mainly into the internal granular layer and dendrites into the external plexiform layer. Outside OB the axons arrived at the anterior olfactory nucleus, primary olfactory cortex, olfactory tubercle, and cortical nucleus of the amygdaloid complex. These fibers appeared to terminate in normal target areas. These findings show that the olfactory system at 5-6 weeks of age still has the capacity to integrate newly migrated neurons and to receive newly growing fibers from the transplant.     

Comparative laminar distribution of various autoradiographic cholinergic markers in adult rat main olfactory bulb

To provide anatomical information on the complex effects of acetylcholine (ACh) in the olfactory bulb (OB), the distribution of different cholinergic muscarinic and nicotinic receptor sub-types was studied by quantitative in vitro autoradiography. The muscarinic M1-like and M2-like sub-types, as well as the nicotinic bungarotoxin-insensitive (alpha 4 beta 2-like) and bungarotoxin-sensitive (alpha 7-like) receptors were visualized using [3H]pirenzepine, [3H]AF-DX 384, [3H]cytisine and [125I] alpha-bungarotoxin (BTX), respectively. In parallel, labelling patterns of [3H]vesamicol (vesicular acetylcholine transport sites) and [3H]hemicholinium-3 (high-affinity choline uptake sites), two putative markers of cholinergic nerve terminals, were investigated. Specific labelling for each cholinergic radioligand is distributed according to a characteristic laminar and regional pattern within the OB revealing the lack of a clear overlap between cholinergic afferents and receptors. The presynaptic markers, [3H]vesamicol and [3H]hemicholinium-3, demonstrated similar laminar pattern of distribution with two strongly labelled bands corresponding to the glomerular layer and the area around the mitral cell layer. Muscarinic M1-like and M2-like receptor sub-types exhibited unique distribution with their highest levels seen in the external plexiform layer (EPL). Intermediate M1-like and M2-like binding densities were found throughout the deeper bulbar layers. In the glomerular layer, the levels of muscarinic receptor subtypes were low, the level of M2-like sites being higher than M1. Both types of nicotinic receptor sub-types displayed distinct distribution pattern. Whereas [125I] alpha-BTX binding sites were mostly concentrated in the superficial bulbar layers, [3H]cytisine binding was found in the glomerular layers, as well as the mitral cell layer and the underlying laminae. An interesting feature of the present study is the visualization of two distinct cholinoceptive glomerular subsets in the posterior OB. The first one exhibited high levels of both [3H]vesamicol and [3H]hemicholinium-3 sites. It corresponds to the previously identified atypical glomeruli and apparently failed to express any of the cholinergic receptors under study. In contrast, the second subset of glomeruli is not enriched with cholinergic nerve terminal markers but displayed high amounts of [3H]cytisine/nicotinic binding sites. Taken together, these results suggest that although muscarinic receptors have been hypothesized to be mostly involved in cholinergic olfactory processing and short-term memory in the OB, nicotinic receptors, especially of the cytisine/ alpha 4 beta 2 sub-type, may have important roles in mediating olfactory transmission of efferent neurons as well as in a subset of olfactory glomeruli.