Neural circuit mechanisms for pattern detection and feature combination in olfactory cortex

Odors are initially encoded in the brain as a set of distinct physicochemical characteristics but are ultimately perceived as a unified sensory object--a "smell." It remains unclear how chemical features encoded by diverse odorant receptors and segregated glomeruli in the main olfactory bulb (MOB) are assembled into integrated cortical representations. Combining patterned optical microstimulation of MOB with in vivo electrophysiological recordings in anterior piriform cortex (PCx), we assessed how cortical neurons decode complex activity patterns distributed across MOB glomeruli. PCx firing was insensitive to single-glomerulus photostimulation. Instead, individual cells reported higher-order combinations of coactive glomeruli resembling odor-evoked sensory maps. Intracellular recordings revealed a corresponding circuit architecture providing each cortical neuron with weak synaptic input from a distinct subpopulation of MOB glomeruli. PCx neurons thus detect specific glomerular ensembles, providing an explicit neural representation of chemical feature combinations that are the hallmark of complex odor stimuli.     

Glycoconjugates in normal human kidney. A histochemical study using 13 biotinylated lectins

The avidin-biotin-peroxidase complex technique was used with 13 lectins to study the glycoconjugates of normal human renal tissue. The evaluated lectins included Triticum vulgaris (WGA), Concanavalin ensiformis (ConA), Phaseolus vulgaris leukoagglutinin and erythroagglutinin (PHA-L and PHA-E), Lens culinaris (LCA), Pisum sativum (PSA), Dolichos biflorus (DBA), Glycine max (SBA), Arachis hypogaea (PNA), Sophora japonica (SJA), Bandeiraea simplicifolia I (BSL-I), Ulex europaeus I (UEA-I) and Ricinus communis I (RCA-I). Characteristic and reproducible staining patterns were observed. WGA and ConA stained all tubules; PHA-L, PHA-E, LCA, PSA stained predominantly proximal tubules; DBA, SBA, PNA, SJA and BSL-I stained predominantly distal portions of nephrons. In glomeruli, WGA and PHA-L stained predominantly visceral epithelial cells; ConA stained predominantly basement membranes and UEA-I stained exclusively endothelial cells. UEA-I also stained endothelial cells of other blood vessels and medullary collecting ducts. Sialidase treatment before staining caused marked changes of the binding patterns of several lectins including a focal loss of glomerular and tubular staining by WGA; an acquired staining of endothelium by PNA and SBA; and of glomeruli by PNA, SBA, PHA-E, LCA, PSA and RCA-I. The known saccharide specificities and binding patterns of the lectins employed in this study allowed some conclusions about the nature and the distribution of the sugar residues in the oligosaccharide chains of renal glycoconjugates. The technique used in this report may be applicable to other studies such as evaluation of normal renal maturation, classification of renal cysts and pathogenesis of nephrotic syndrome. The observations herein reported may serve as a reference for these studies.     

Lectin histochemical study of olfactory neurons in the eel

Lectin histochemical studies were performed on paraffin embedded sections of the olfactory system of the eel to identify specific glycoconjugates on the surface of primary olfactory neurons. The olfactory receptors, the olfactory nerve fibres and their terminals in the bulbs were labelled with the lectins (SBA, BSA-I, BSA-I-B4 and DBA) HRP-conjugated or biotinylated. The lectin staining patterns indicate that the membrane of olfactory neurons of the eel had oligosaccharides with alpha-galactose and alpha-N-acetyl-D-galactosamine residues. These findings represent the demonstration of a molecular probe that recognizes specific sets of neurons. The identical histochemical features previously described in the olfactory neurons in amphibians suggest that these carbohydrate moieties might to related to modulation of the cell-cell interactions in the olfactory system of vertebrates.     

Heterogeneity in olfactory neurons in mouse revealed by differential expression of glycoconjugates

Cell surface glycoconjugates have been implicated in the growth and guidance of subpopulations of primary olfactory axons. While subpopulations of primary olfactory neurons have been identified by differential expression of carbohydrates in the rat there are few reports of similar subpopulations in the mouse. We have examined the spatiotemporal expression pattern of glycoconjugates recognized by the lectin from Wisteria floribunda (WFA) in the mouse olfactory system. In the developing olfactory neuroepithelium lining the nasal cavity, WFA stained a subpopulation of primary olfactory neurons and the fascicles of axons projecting to the target tissue, the olfactory bulb. Within the developing olfactory bulb, WFA stained the synaptic neuropil of the glomerular and external plexiform layers. In adults, strong expression of WFA ligands was observed in second-order olfactory neurons as well as in neurons in several higher order olfactory processing centres in the brain. Similar, although distinct, staining of neurons in the olfactory pathway was detected with Dolichos biflorus agglutinin. These results demonstrate that unique subpopulations of olfactory neurons are chemically coded by the expression of glycoconjugates. The conserved expression of these carbohydrates across species suggests they play an important role in the functional organization of this region of the nervous system.     

Glycoconjugates in normal human kidney

Summary The avidin-biotin-peroxidase complex technique was used with 13 lectins to study the glycoconjugates of normal human renal tissue. The evaluated lectins included Triticum vulgaris (WGA), Concanavalin ensiformis (ConA), Phaseolus vulgaris leukoagglutinin and erythroagglutinin (PHA-L and PHA-E), Lens culinaris (LCA), Pisum sativum (PSA), Dolichos biflorus (DBA), Glycine max (SBA), Bandeiraea simplicifolia I (BSL-I), Ulex europaeus I (UEA-I) and Ricinus communis I (RCA-I). Characteristic and reproducible staining patterns were observed. WGA and ConA stained all tubules; PHA-L, PHA-E, LCA, PSA stained predominantly proximal tubules; DBA, SBA, PNA, SJA and BSL-I stained predominantly distal portions of nephrons. In glomeruli, WGA and PHA-L stained predominantly visceral epithelial cells; ConA stained predominantly basement membranes and UEA-I stained exclusively endothelial cells. UEA-I also stained endothelial cells of other blood vessels and medullary collecting ducts. Sialidase treatment before staining caused marked changes of the binding patterns of several lectins including a focal loss of glomerular and tubular staining by WGA; an acquired staining of endothelium by PNA and SBA; and of glomeruli by PNA, SBA, PHA-E, LCA, PSA and RCA-I. The known saccharide specificities and binding patterns of the lectins employed in this study allowed some conclusions about the nature and the distribution of the sugar residues in the oligosaccharide chains of renal glycoconjugates. The technique used in this report may be applicable to other studies such as evaluation of normal renal maturation, classification of renal cysts and pathogenesis of nephrotic syndrome. The observations herein reported may serve as a reference for these studies.     

In vivo imaging of neuronal activity by targeted expression of a genetically encoded probe in the mouse

Genetically encoded probes show great promise in permitting functional imaging of specified neuronal populations in the intact nervous system, yet their in vivo application has been limited. Here, we have targeted expression of synapto-pHluorin, a pH-sensitive protein that reports synaptic vesicle fusion, to olfactory sensory neurons in mouse. Synapto-pHluorin selectively labeled presynaptic terminals of sensory neurons in glomeruli of the olfactory bulb. Odorant stimulation evoked large-amplitude fluorescence increases that were localized to individual glomeruli in vivo, correlated with presynaptic calcium influx, graded with stimulus intensity, and stable over a period of days. Spatial patterns of odorant-activated glomeruli were distributed and did not change systematically with increasing carbon chain length, in contrast to the finely organized chemotopy that has been reported using other imaging methods. Targeted expression of synapto-pHluorin in mouse will permit the analysis of previously inaccessible neuronal populations and chronic imaging from genetically identified neurons in vivo.     

Glomerular sialoconjugates of developing and mature rat kidneys

The appearance of sialoconjugates in developing rat kidney glomeruli was studied using lectins and neuraminidase-lectin staining sequences. In the early S-shaped bodies, binding of Maclura pomifera (MPA; specific for galactosaminyl residues of glycoconjugates) could be detected in the presumptive podocyte layer at the apex of these cells, but notably no binding of lectins specific for sialic acid could be seen. During further morphologic maturation of the S-shaped bodies, binding of Limax flavus (LFA; specific for sialic acids) and Triticum vulgaris (WGA; specific for sialic acids and N-acetyl glucosaminyl moieties) appeared at the apex of podocytes and extended subsequently along the lateral membranes to the base of these cells. In morphologically mature glomeruli, LFA stained not only the base of podocytes but also glomerular basement membranes. WGA and MPA bound to the capillary endothelia as well as to the structures bound by LFA. The intensity of WGA binding increased considerably after 5 days of postnatal life, seemingly in parallel with the decrease and ultimate disappearance of MPA binding. In addition to showing individual appearance pattern for various lectin binding sites, these studies give evidence of previously unrecognized postnatal completion of the components of glomerular filtration barrier.     

Precise circuitry links bilaterally symmetric olfactory maps

Olfactory sensory neurons expressing a common receptor gene converge onto one or a few glomeruli with stereotyped positions within the mouse main olfactory bulb (MOB), producing a map of approximately 1800 olfactory columns representing approximately 1000 odorant receptors. Here, we report that this precise olfactory map is maintained over several synapses that ultimately cross MOB hemispheres to link bilateral isofunctional olfactory columns. Focal injection of tracer into genetically identified glomeruli revealed an exquisite topography that involves a bilateral connection via the anterior olfactory nucleus pars externa (AONpE) that links isofunctional olfactory columns in the contralateral MOB. Physiological and behavioral assays revealed an important role for the AONpE in bilateral exchange of odorant-specific information. These results indicate that the interbulbar link through the AONpE integrates bilateral olfactory sensory maps and exchanges olfactory information, positioning it as a unique model system for studying interhemispheric connections.     

Heterogeneous Sensory Innervation and Extensive Intrabulbar Connections of Olfactory Necklace Glomeruli

The mammalian nose employs several olfactory subsystems to recognize and transduce diverse chemosensory stimuli. These subsystems differ in their anatomical position within the nasal cavity, their targets in the olfactory forebrain, and the transduction mechanisms they employ. Here we report that they can also differ in the strategies they use for stimulus coding. Necklace glomeruli are the sole main olfactory bulb (MOB) targets of an olfactory sensory neuron (OSN) subpopulation distinguished by its expression of the receptor guanylyl cyclase GC-D and the phosphodiesterase PDE2, and by its chemosensitivity to the natriuretic peptides uroguanylin and guanylin and the gas CO₂. In stark contrast to the homogeneous sensory innervation of canonical MOB glomeruli from OSNs expressing the same odorant receptor (OR), we find that each necklace glomerulus of the mouse receives heterogeneous innervation from at least two distinct sensory neuron populations: one expressing GC-D and PDE2, the other expressing olfactory marker protein. In the main olfactory system it is thought that odor identity is encoded by a combinatorial strategy and represented in the MOB by a pattern of glomerular activation. This combinatorial coding scheme requires functionally homogeneous sensory inputs to individual glomeruli by OSNs expressing the same OR and displaying uniform stimulus selectivity; thus, activity in each glomerulus reflects the stimulation of a single OSN type. The heterogeneous sensory innervation of individual necklace glomeruli by multiple, functionally distinct, OSN subtypes precludes a similar combinatorial coding strategy in this olfactory subsystem.     

Lectin binding in human skeletal muscle: a comparison of 15 different lectins

Summary Fifteen lectin-horseradish peroxidase conjugates have been used in a comprehensive histochemical study of human skeletal muscle. The staining patterns of many lectins were found to be coincident with the known distributions of types I, III, IV and V collagen, fibronectin and laminin. One lectin,Bandeiraea simplicifolia (BSA I), selectively stained capillaries in a blood group-specific manner, the significance of which is unknown. The results show that although lectins are useful cytochemical probes for identifying tissue glycoconjugates, lectin binding is not solely determined by monosaccharide specificity as lectins which interact with the same sugars may have completely different staining patterns. Factors such as accessibility, glycan conformation and oligosaccharide sequence also affect lectin binding in tissues. For these reasons, we conclude that a comprehensive histochemical investigation of tissue glycoconjugates should employ a large number of lectins, preferably with overlapping sugar specificities.     

Glycoconjugates are stage- and position-specific cell surface molecules in the developing olfactory system, 2: Unique carbohydrate antigens are topographic markers for selective projection patterns of olfactory axons.

Three monoclonal antibodies specific for different carbohydrate antigens were used to analyze the development of the olfactory system in rats. CC2 antibodies react with a subset of main olfactory neurons, their axons, and terminals in the olfactory bulb. CC2 antigens are expressed on dorsomedial neurons in the olfactory epithelium (OE) from embryonic (E) day 15 to adults. In the olfactory bulb (OB), only dorsomedially located glomeruli express CC2 glycoconjugates from postnatal day (P) 2 to adults. Thus CC2 defines a dorsomedially organized projection that is established early in embryonic development and continues in adults. P-Path antibodies react with antigens that are expressed on the olfactory nerve in embryos, and are also detected on cell bodies in the neuroepithelium and in glomeruli of the OB at P2. At P14, P-Path staining is weaker, but remains present on many cells in the epithelium and in many glomeruli in the bulb. Postnatally, P-Path immunostaining continues to decrease in most regions of the OE and OB. At P35 and afterwards, only a few P-Path-positive neuronal cells can be detected in the OE. Furthermore, after P35 only two groups of glomeruli in the OB are P-Path immunoreactive. One is situated adjacent to the accessory olfactory bulb (AOB) at the dorsocaudal surface of the OB. The other is adjacent to the AOB at the ventrocaudal surface of the OB. Thus, in adults, P-Path glycoconjugates are expressed in neurons and axons that project only to a specific subset of caudal glomeruli of the OB. Monoclonal antibody 1B2, reacts with beta-galactose-terminating glycolipids and glycoproteins. At P2, 1B2 immunoreactivity is seen on a subset of cell bodies that are distributed throughout the OE and is expressed in most glomeruli in the OB at this age. By P35 and in adults, 1B2 continues to be expressed on a subset of neurons in the OE that project to only a small subset of glomeruli in the OB. Unlike CC2 and P-Path antigens that define specific groups of glomeruli, 1B2-immunoreactive glomeruli do not have a detectable spatial pattern. It is more likely that 1B2 antigens define a specific stage in the maturation of connections between the OE and OB.     

Glycoconjugates are stage- and position-specific cell surface molecules in the developing olfactory system, 2: Unique carbohydrate antigens are topographic markers for selective projection patterns of olfactory axons

Three monoclonal antibodies specific for different carbohydrate antigens were used to analyze the development of the olfactory system in rats. CC2 antibodies react with a subset of main olfactory neurons, their axons, and terminals in the olfactory bulb. CC2 antigens are expressed on dorsomedial neurons in the olfactory epithelium (OE) from embryonic (E) day 15 to adults. In the olfactory bulb (OB), only dorsomedially located glomeruli express CC2 glycoconjugates from postnatal day (P) 2 to adults. Thus CC2 defines a dorsomedially organized projection that is established early in embryonic development and continues in adults. P-Path antibodies react with antigens that are expressed on the olfactory nerve in embryos, and are also detected on cell bodies in the neuroepithelium and in glomeruli of the OB at P2. At P14, P-Path staining is weaker, but remains present on many cells in the epithelium and in many glomeruli in the bulb. Postnatally, P-Path immunostaining continues to decrease in most regions of the OE and OB. At P35 and afterwards, only a few P-Path-positive neuronal cells can be detected in the OE. Furthermore, after P35 only two groups of glomeruli in the OB are P-Path immunoreactive. One is situated adjacent to the accessory olfactory bulb (AOB) at the dorsocaudal surface of the OB. The other is adjacent to the AOB at the ventrocaudal surface of the OB. Thus, in adults, P-Path glycoconjugates are expressed in neurons and axons that project only to a specific subset of caudal glomeruli of the OB. Monoclonal antibody 1B2, reacts with β-galactose-terminating glycolipids and glycoproteins. At P2, 1B2 immunoreactivity is seen on a subset of cell bodies that are distributed throughout the OE and is expressed in most glomeruli in the OB at this age. By P35 and in adults, 1B2 continues to be expressed on a subset of neurons in the OE that project to only a small subset of glomeruli in the OB. Unlike CC2 and P-Path antigens that define specific groups of glomeruli, 1B2-immunoreactive glomeruli do not have a detectable spatial pattern. It is more likely that 1B2 antigens define a specific stage in the maturation of connections between the OE and OB.     

Lectin-binding patterns in the olfactory system of the lizard, Physignathus lesueurii

Lectin binding histochemistry was performed on the olfactory system of Physignathus lesueurii to investigate the distribution and density of defined carbohydrate terminals on the cell-surface glycoproteins of the olfactory and vomeronasal receptor cells and their terminals in the olfactory bulbs. The lectin staining patterns indicate that the vomeronasal and olfactory receptor cells are characterized by glycoconjugates containing alpha-D-galactose and N-acetyl-D-glucosamine terminal residues. The presence of specific glycoproteins, whose terminal sugars are detected by lectin binding, might be related to the chemoreception and transduction of the odorous message into a nervous signal or to the histogenesis and development of the olfactory system. The olfactory and vomeronasal receptor cells are vertebrate neurons that undergo a continual cycle of proliferation not only during development but also in mature animals.     

Regional differences in the distribution of endogenous receptors for carbohydrate constituents of cellular glycoconjugates, especially lectins, in cortex, hippocampus, basal ganglia and thalamus of adult human brain

Ten different types of labelled neoglycoproteins, exposing glycohistochemically pivotal carbohydrate moieties that mostly are constituents of naturally occurring glycoconjugates with an aromatic spacer, were synthesized. The panel was applied to fixed, paraffin-embedded sections of different cortical regions and white matter, of hippocampal gyrus, basal ganglia, thalamus nuclei and adjacent areas of adult human brain to comprehensively map the presence of respective binding sites in these parts. Compliance with accepted criteria for specificity of binding was routinely ascertained. Overall, not a uniform binding pattern, but a distinct distribution with regional differences on the level of specific cytoplasmic and nuclear staining in nerve cells was determined, fiber structures being generally labelled with medium or strong intensity. For example, among the neurons localized in the five cortical laminae the binding of N-acetyl-D-galactosamine varied from strong to undetectable. Biochemical analysis, employing carbohydrate residues as affinity ligands in chromatography, proved that the neuroanatomically different regions exhibited a pattern of receptors with notable similarities. These results on endogenous binding sites for glycoconjugates, especially lectins, are complementary to assessment of localization of cellular glycoconjugates by plant lectins and carbohydrate-specific monoclonal antibodies. They are thus a further obligatory step to substantiate the physiological roles of recognitive protein-carbohydrate interactions in the central nervous system.     

Binding Patterns of Lectins with GalNAc specificity in the mouse dorsal root ganglia and spinal cord

To localize membrane glycoconjugates in neurons of the mouse spinal cord and dorsal root ganglia (DRG), cryostat sections of newborn (P0), 7 day-old (P7), P14, P21 and P31 animals were stained with ten FITC-conjugated plant lectins, the majority of them recognizing N-acetyl-D-galactosamine (GalNAc) terminal sugar residues. In the dorsal root ganglia of P0 animals, the different lectins showed distinct patterns of labeling in either cells of the nervous system, including neurons, or other structures such as nerves or blood vessels. Moreover, some of these lectins showed important changes in their pattern of labeling during postnatal development. This was especially relevant for lectins that label a subpopulation of small-sized cells that have been previously identified as the nociceptive cells of the DRG. Enzymatic digestion of sections with neuraminidase removes sialic acid from the carbohydrate chains of glycoconjugates thus exposing novel sugar residues. When this treatment was applied to DRG sections from postnatal animals the pattern of lectin staining was either changed or eliminated and heterogeneous subsets of glycoconjugates normally masked by this sugar were exposed. In the spinal cord of PO animals, none of the lectins labeled cells in the central gray matter. However, after the enzymatic digestion of sections with neuraminidase, spinal cord motoneurons and some other cells were labeled by two of the lectins suggesting that GalNAc residues present in these cells are normally masked by terminal sialic acid. Altogether, these results show important changes in the temporal and spatial expression of glycoconjugates that may be relevant for the postnatal development of the CNS and PNS of mice.     

Regional differences in the distribution of endogenous receptors for carbohydrate constituents of cellular glycoconjugates,especially lectins,in cortex,hippocampus, basal ganglia and thalamus of adult human brain

Summary Ten different types of labelled neoglycoproteins, exposing glycohistochemically pivotal carbohydrate moieties that mostly are constituents of naturally occurring glycoconjugates with an aromatic spacer, were synthesized. The panel was applied to fixed, paraffin-embedded sections of different cortical regions and white matter, of hippocampal gyrus, basal ganglia, thalamus nuclei and adjacent areas of adult human brain to comprehensively map the presence of respective binding sites in these parts. Compliance with accepted criteria for specificity of binding was routinely ascertained. Overall, not a uniform binding pattern, but a distinct distribution with regional differences on the level of specific cytoplasmic and nuclear staining in nerve cells was determined, fiber structures being generally labelled with medium or strong intensity. For example, among the neurons localized in the five cortical laminae the binding of N-acetyl-d-galactosamine varied from strong to undetectable. Biochemical analysis, employing carbohydrate residues as affinity ligands in chromatography, proved that the neuroanatomically different regions exhibited a pattern of receptors with notable similarities. These results on endogenous binding sites for glycoconjugates, especially lectins, are complementary to assessment of localization of cellular glycoconjugates by plant lectins and carbohydrate-specific monoclonal antibodies. They are thus a further obligatory step to substantiate the physiological roles of recognitive protein-carbohydrate interactions in the central nervous system.     

Neuropilin-2 mediates axonal fasciculation, zonal segregation, but not axonal convergence, of primary accessory olfactory neurons

The mechanisms that underlie axonal pathfinding of vomeronasal neurons from the vomeronasal organ (VNO) in the periphery to select glomeruli in the accessory olfactory bulb (AOB) are not well understood. Neuropilin-2, a receptor for secreted semaphorins, is expressed in V1R- and V3R-expressing, but not V2R-expressing, postnatal vomeronasal neurons. Analysis of the vomeronasal nerve in neuropilin-2 (npn-2) mutant mice reveals pathfinding defects at multiple choice points. Vomeronasal sensory axons are severely defasciculated and a subset innervates the main olfactory bulb (MOB). While most axons of V1R-expressing neurons reach the AOB and converge into distinct glomeruli in stereotypic locations, they are no longer restricted to their normal anterior AOB target zone. Thus, Npn-2 and candidate pheromone receptors play distinct and complementary roles in promoting the wiring and patterning of sensory neurons in the accessory olfactory system.     

A dot blot technique for the analysis of interactions of lectins with glycosaminoglycans

Summary Glycosaminoglycans are polysaccharides which are widely distributed throughout connective tissue, where they form an essential part of the extracellular matrix. Connective tissue is often stained by lectins, and it is not known whether this staining is due to the interaction of lectins with the glycosaminoglycans or due to the binding of lectins to other glycoconjugates within the matrix. A dot blot technique is presented by which the interaction of lectins with glycosaminoglycans can be analysed.     

Convergence in the piriform cortex

How are the responses to distinct chemical features integrated to form an olfactory perceptual object? In this issue of Neuron, Davison and Ehlers show that individual piriform cortex neurons receive convergent input from up to 10% of main olfactory bulb glomeruli and are activated by specific spatial patterns of coactive glomeruli.     

Offset response of the olfactory projection neurons in the moth antennal lobe

We investigated a population activity of central olfactory neurons after the termination of odor input. Olfactory response of projection neurons in the moth primary olfactory center was characterized using in vivo intracellular recording and staining techniques. The population activity changed rapidly to the different states after the stimulus offset. The response after stimulus offset represents information regarding odor identity. We analyzed the spatial distribution of offset-activated glomeruli in a virtual neuronal population that was reconstructed using accumulated individual recordings obtained from different specimens. The offset-activated glomeruli tended to be widely distributed, whereas the onset-activated glomeruli were relatively clustered. These results suggest the importance of lateral interaction in shaping the offset olfactory response.