Development and further characterization of a small subclass of rat olfactory receptor neurons that shows immunoreactivity for the HSP70 heat shock protein

We previously described a rat olfactory receptor neuron (ORN) subpopulation [the 2A4(+) ORNs] that shows uniquely strong reactivity with antibodies to the 70-kD heat shock protein (HSP70) family of molecular chaperones (Carr et al. [1994] J. Comp. Neurol. 348:150-160). The 2A4(+)ORNs are dispersed through zones II-IV of the olfactory epithelium (OE), and their axons project to only two or three glomeruli that are located consistently in each olfactory bulb (OB). To date, the 2A4(+)ORN subpopulation is the only cell population to show such distinct HSP70 immunoreactivity as well as the most discrete ORN subpopulation to be so labeled. The present report shows that 2A4(+)ORN neurons first appear between postnatal days 7 (P7) and P10. Initially, low cell numbers rise to a density of 0.1 2A4(+)ORNs/mm OE length by P14, plateau at 0.9 2A4(+)ORNs/mm by P49, then fall to adult values of 0.4 cells/mm. Autoradiographic birthdating indicates that almost all of these early appearing 2A4(+)ORNs are generated postnatally, in contrast to the prenatal generation of all ORN subpopulations characterized to date by their expression of olfactory receptor protein mRNAs. A developmentally related increase in the mean depth of 2A4(+)ORNs within the OE also occurs. In the OB, initial 2A4(+)axonal projections are to only two or three glomeruli, as in adults. Slight but significant rostral shifts in (+)glomerular location occur with development. The 2A4(+)ORN immunoreactivity was found to be due to expression of HSP70, the dominant stress-inducible member of the HSP70 family, rather than constitutively expressed HSC70. In addition, despite their presence in rat OE, no 2A4(+)ORNs were found in mice, gerbils, guinea pigs, or hamsters.     

Odor-induced, activity-dependent transneuronal gene induction in vitro: mediation by NMDA receptors

Expression of tyrosine hydroxylase (TH) by juxtaglomerular (JG) neurons of the olfactory bulb (OB) requires innervation of the bulb by olfactory receptor neurons (ORNs). ORN lesion selectively downregulates TH in JG neurons. In reversible odor deprivation, TH expression is downregulated as the naris is closed and then upregulated upon naris reopening. The mechanism or mechanisms regulating this dependence are unknown. TH expression could be regulated by trophic factor release and/or synaptic activity from ORN terminals. We investigated TH expression in cocultures of dissociated postnatal rat OB cells and embryonic olfactory neuroepithelium (OE) slice explants. TH-positive neurons in control dissociated OB cell cultures alone comprise only a small fraction of the total population of cells present in the culture. However, when OE slice explants are cocultured with dispersed OB cells, there is a mean 2.4-fold increase in the number of TH-positive neurons. ORNs in vivo use glutamate as a neurotransmitter. Broad spectrum excitatory amino acid antagonists (kyurenic acid) or selective antagonists of the NMDA receptor (APV) both prevent induction of TH expression in OE-OB cocultures. Furthermore, pulse application of NMDA stimulates TH expression in OB neurons in the absence of OE. In vitro, OB TH neurons express NMDA receptors, suggesting that NMDA stimulation is acting directly on TH neurons. Exposure of OE explants to natural odorants results in upregulation of TH, presumably through increased ORN activity, which could be blocked by APV. These findings indicate that odorant-stimulated glutamate release by ORN terminals regulates TH expression via NMDA receptors on JG dopaminergic neurons.     

Odorants differentially enhance phosphoinositide turnover and adenylyl cyclase in olfactory receptor neuronal cultures

Both the cAMP and the phosphoinositide (PI) second messenger systems have been implicated in olfactory signal transduction. We have developed a primary culture system of mammalian olfactory receptor neurons (ORNs; Ronnett et al., 1991a) to permit analysis of odorant-induced second messenger system activation in the intact ORN. The ability of a series of odorants to stimulate PI turnover and adenylyl cyclase was examined. All odorants stimulated both second messenger systems, although with differential potencies. Stimulation of PI turnover desensitized upon reexposure of cultures to odorant. The enhancement by single odorants of both adenylyl cyclase and PI turnover, but to varying degrees, affords a mechanism for increased specificity in olfactory signal transduction.     

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.     

Nitric oxide mediates the formation of synaptic connections in developing and regenerating olfactory receptor neurons

Nitric oxide (NO) is a diffusible free radical that functions as a second messenger and neurotransmitter. NO synthase (NOS) is highly and transiently expressed in neurons of the developing olfactory epithelium during migration and establishment of primary synapses in the olfactory bulb. NOS is first expressed at E11 in cells of the presumptive nervous layer of the olfactory placode. NOS immunoreactivity persists in the descendants of these cells that differentiate into embryonic olfactory receptor neurons (ORNs). Olfactory NOS expression in the ORN and in its afferents rapidly declines after birth and is undetectable by P7. Following bulbectomy, NOS expression is rapidly induced in the regenerating ORN and is particularly enriched in their outgrowing axons. Immunoblot and Northern blot analyses similarly demonstrate an induction of NOS protein and mRNA expression, respectively, the highest levels of which coincide with peaks of ORN regeneration. These data argue against a role for NO in odorant-sensitive signal transduction, but suggest a prominent function for NO in activity-dependent establishment of connections in both developing and regenerating olfactory neurons.     

Identification of olfactory receptor mRNA sequences from the rat olfactory bulb glomerular layer

In situ hybridization has demonstrated mRNA for olfactory receptors (OR) in the axon terminals of olfactory receptor neurons. Neurons that express the same OR appear to send their axons to two stereotyped glomeruli in the olfactory bulb (OB). Based on these observations, we tested the feasibility of using RT-PCR to isolate and sequence OR mRNA from small samples of the rat OB glomerular layer. Biomagnetic mRNA isolation followed by RT-PCR yielded partial sequences for 21 novel members of the OR family. The results suggest that the topography of OR mRNA can be mapped across the OB, to study synaptic specificity and odor representation in the olfactory system.     

Effects of fenvalerate on the development of olfactory perception in a beetle

Low doses of fenvalerate (a Type II pyrethroid) were applied to the beetle Tenebrio molitor at pupation, to ascertain its effects on the developing olfactory system. Doses of fenvalerate that prevent the formation of glomeruli in the primary olfactory neuropil (antennal lobes) also inhibit olfactory orientation behavior for different odors, despite the fact that sensory neurons developed responses to these odors. Even when lower amounts of fenvalerate that allowed glomeruli to develop were applied to pupae, the olfactory behavior was affected. Therefore, the formation of glomerular structures within the antennal lobe is not sufficient to establish olfactory behavior. A possible reason for this developmental effect of fenvalerate is a change in the odotopic arrangement of sensory axons within the glomeruli.     

Marked restoration of density and total number of mature (knob-bearing) olfactory receptor neurons in rats recovering from early hypothyroid-induced growth retardation

Our recent studies have shown that restoration of thyroid function in developing hypothyroid rats results in upregulation of olfactory neurogenesis and compensatory proliferation of olfactory receptor neurons (ORN) in the olfactory epithelium (OE) (Paternostro and Meisami, Dev. Brain Res., 76 (1993) 151-161; ibid., 83 (1994) 151-162). It was not clear, however, whether the newly forming ORNs undergo complete maturational stages. To determine the effects of restoration of thyroid function on maturation of ORNs, the density and total number of mature ORNs were estimated in the OE of euthyroid and hypothyroid rats at postnatal days 1, 12, 25 and 90 and the results were compared with those in rats allowed to recover from early thyroid deficiency at weaning (day 25). As a marker for mature ORNs, and on the basis of one olfactory dendritic knob per ORN, the density and total number of the olfactory knobs were determined in the entire extent of the OE covering the nasal septum. Hypothyroidism was induced by adding propylthiouracil (PTU) to the drinking water (1 g/l) from birth until days 12, 25 or 90 of age. Recovery from hypothyroidism was induced by withdrawal of PTU at day 25, leading to restoration of thyroid function and somatic growth recovery. The density of olfactory knobs was determined in 1 microm semi-thin sections stained with toluidine blue. In the normal rats, the number of olfactory knobs (= mature ORNs) increased 8.5- and 3-fold during postnatal days 1-25 and 25-90 respectively, reaching a mean value of 4 X 10(6)/septal OE, compared to 2.8- and 1.4-fold, respectively, for the hypothyroid rats. This led to deficits of 51% and 76% in the number of mature ORNs in the 25- and 90-day-old hypothyroid rats. In rats allowed to recover, the number of mature ORNs increased 4.5-fold during postnatal days 25-90 (3 X > hypothyroid rats and 1.5 X > controls). The results indicate marked upregulation of the maturational process of the ORNs and their compensatory accretion within the OE of the recovery group. The recovery process was not complete however, as indicated by a remaining deficit of about 25% in the total number of mature ORN, compared to normal 90-day controls. Thus thyroid hormones are essential for accretion of new mature ORNs in both the suckling and postweaning rats. Also, the ORNs show a remarkable ability to recover from severe early hypothyroid-induced growth retardation and attain normal mature state.     

A subset of olfactory neurons that selectively express cGMP-stimulated phosphodiesterase (PDE2) and guanylyl cyclase-D define a unique olfactory signal transduction pathway

Odorant information is encoded by a series of intracellular signal transduction events thought to be mediated primarily by the second messenger cAMP. We have found a subset of olfactory neurons that express the cGMP-stimulated phosphodiesterase (PDE2) and guanylyl cyclase-D (GC-D), suggesting that cGMP in these neurons also can have an important regulatory function in olfactory signaling. PDE2 and GC-D are both expressed in olfactory cilia where odorant signaling is initiated; however, only PDE2 is expressed in axons. In contrast to most other olfactory neurons, these neurons appear to project to a distinct group of glomeruli in the olfactory bulb that are similar to the subset that have been termed "necklace glomeruli." Furthermore, this subset of neurons are unique in that they do not contain several of the previously identified components of olfactory signal transduction cascades involving cAMP and calcium, including a calcium/calmodulin-dependent PDE (PDE1C2), adenylyl cyclase III, and cAMP-specific PDE (PDE4A). Interestingly, these latter three proteins are expressed in the same neurons; however, their subcellular distribution is distinct. PDE1C2 and adenylyl cyclase III are expressed almost exclusively in the olfactory cilia whereas PDE4A is present only in the cell bodies and axons. These data strongly suggest that selective compartmentalization of different PDEs and cyclases is an important feature for the regulation of signal transduction in olfactory neurons and likely in other neurons as well. In addition, the data implies that an olfactory signal transduction pathway specifically modulated by cGMP is present in some neurons of the olfactory neuroepithelium.     

2-Mercaptoethanol is a survival factor for olfactory, cortical and hippocampal neurons in short-term dissociated cell culture

A medium originally designed for lymphocyte growth promoted robust survival of olfactory receptor neurons (ORNs) in short-term (4-day), dissociated cell culture. The key ingredient for survival of neurons in both serum and serum-free conditions was 2-mercaptoethanol (2-ME). Enhancement of survival may be thiol-mediated because two other thiol compounds, 2-mercaptoethylamine and monothioglycerol, also increased ORN survival. Addition of 2-ME also significantly increased survival of embryonic cortical and hippocampal neurons in a serum-free medium, and embryonic cortical neurons in a serum-containing medium. After plating and growth in a serum-free medium containing 2-ME, survival of all three types of neurons was equivalent to, or greater than, survival in serum-containing media. Thus, thiols such as 2-ME promote the survival of multiple types of neurons in short-term cell culture.     

How synchronization properties among second-order sensory neurons can mediate stimulus salience.

Spatial patterns of glomerular activity in the vertebrate olfactory bulb and arthropod antennal lobe reflect an important component of first-order olfactory representation and contribute to odorant identification. Higher concentration odor stimuli evoke broader glomerular activation patterns, resulting in greater spatial overlap among different odor representations. However, behavioral studies demonstrate results contrary to what these data might suggest: Honeybees are more, not less, able to discriminate among odorants applied at higher concentrations. Using a computational model of the honeybee antennal lobe, the authors show that changes in synchronization patterns among antennal lobe projection neurons, as observed electrophysiologically, could parsimoniously underlie these observations. The results suggest that stimulus salience, as defined behaviorally, is directly correlated with the degree of synchronization among second-order olfactory neurons. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

CSF levels of HVA and 5-HIAA in drug-free schizophrenic patients and healthy controls: a prospective study focused on their predictive value for outcome in schizophrenia

Recent progress in the studies of the olfactory system, especially in the molecular biological studies, makes it one of the useful sensory model systems for understanding neural mechanisms for the information processing. In the olfactory bulb, the primary center of the olfactory system, glomeruli are regarded as important functional units in the transmission of odorant signals and in processing the olfactory information, but have been believed to be composed by only a small number of neuronal types and thus to be simple in their neuronal and synaptic organization. However, accumulating morphological data reveal that each type of neurons might further consist of several different subpopulations, indicating that the organization of glomeruli might not be so simple as it was believed. Here we describe an aspect of the structural organization of glomeruli, focusing on the heterogeneities of periglomerular neurons in mammalian main olfactory bulb.     

An olfactory sensory map develops in the absence of normal projection neurons or GABAergic interneurons

Olfactory sensory neurons expressing a given odorant receptor project to two topographically fixed glomeruli in the olfactory bulb. We have examined the contribution of different cell types in the olfactory bulb to the establishment of this topographic map. Mice with a homozygous deficiency in Tbr-1 lack most projection neurons, whereas mice with a homozygous deficiency in Dlx-1 and Dlx-2 lack most GABAergic interneurons. Mice bearing a P2-IRES-tau-lacZ allele and deficient in either Tbr-1 or Dlx-1/Dlx-2 reveal the convergence of axons to one medial and one lateral site at positions analogous to those observed in wild-type mice. These observations suggest that the establishment of a topographic map is not dependent upon cues provided by, or synapse formation with, the major neuronal cell types in the olfactory bulb.     

A modified coaxial compound micropipette for extracellular iontophoresis and intracellular recording: fabrication, performance and theory

Different olfactory cues elicit distinct behaviors such as attraction, avoidance, feeding, or mating. In the nematode C. elegans, these cues are sensed by a small number of olfactory neurons, each of which expresses several different odorant receptors. The type of behavioral response elicited by an odorant could be specified by the olfactory receptor or by the olfactory neuron in which the receptor is activated. The attractive odorant diacetyl is detected by the receptor protein ODR-10, which is normally expressed in the AWA olfactory neurons. The repulsive odorant 2-nonanone is detected by the AWB olfactory neurons. Transgenic animals that express ODR-10 in AWB rather than AWA avoid diacetyl, while maintaining qualitatively normal responses to other attractive and repulsive odorants. Animals that express ODR-10 simultaneously in AWA and AWB have a defective response to diacetyl, possibly because of conflicting olfactory inputs. Thus, an animal's preference for an odor is defined by the sensory neurons that express a given odorant receptor molecule.     

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.     

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.     

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.     

Transduction mechanisms in vertebrate olfactory receptor cells

Considerable progress has been made in the understanding of transduction mechanisms in olfactory receptor neurons (ORNs) over the last decade. Odorants pass through a mucus interface before binding to odorant receptors (ORs). The molecular structure of many ORs is now known. They belong to the large class of G protein-coupled receptors with seven transmembrane domains. Binding of an odorant to an OR triggers the activation of second messenger cascades. One second messenger pathway in particular has been extensively studied; the receptor activates, via the G protein Golf, an adenylyl cyclase, resulting in an increase in adenosine 3',5'-cyclic monophosphate (cAMP), which elicits opening of cation channels directly gated by cAMP. Under physiological conditions, Ca2+ has the highest permeability through this channel, and the increase in intracellular Ca2+ concentration activates a Cl- current which, owing to an elevated reversal potential for Cl-, depolarizes the olfactory neuron. The receptor potential finally leads to the generation of action potentials conveying the chemosensory information to the olfactory bulb. Although much less studied, other transduction pathways appear to exist, some of which seem to involve the odorant-induced formation of inositol polyphosphates as well as Ca2+ and/or inositol polyphosphate -activated cation channels. In addition, there is evidence for odorant-modulated K+ and Cl- conductances. Finally, in some species, ORNs can be inhibited by certain odorants. This paper presents a comprehensive review of the biophysical and electrophysiological evidence regarding the transduction processes as well as subsequent signal processing and spike generation in ORNs.     

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.