An isoform of the rod photoreceptor cyclic nucleotide-gated channel β subunit expressed in olfactory neurons

Sensory transduction in olfactory neurons involves the activation of a cyclic nucleotide-gated (CNG) channel by cAMP. Previous studies identified a CNG channel α subunit (CNG2) and a β subunit (CNG5), which when heterologously expressed form a channel with properties similar but not identical to those of native olfactory neurons. We have cloned a new type of CNG channel β subunit (CNG4.3) from rat olfactory epithelium. CNG4.3 derives from the same gene as the rod photoreceptor β subunit (CNG4.1) but lacks the long, glutamic acid-rich domain found in the N terminus of CNG4.1. Northern blot and in situ hybridization revealed that CNG4.3 is expressed specifically in olfactory neurons. Expression of CNG4.3 in human embryonic kidney 293 cells did not lead to detectable currents. Coexpression of CNG4.3 with CNG2 induced a current with significantly increased sensitivity for cAMP whereas cGMP affinity was not altered. Additionally, CNG4.3 weakened the outward rectification of the current in the presence of extracellular Ca2+, decreased the relative permeability for Ca2+, and enhanced the sensitivity for l-cis diltiazem. Upon coexpression of CNG2, CNG4.3, and CNG5, a conductance with a cAMP sensitivity greater than that of either the CNG2/CNG4.3 or the CNG2/CNG5 channel and near that of native olfactory channel was observed. Our data suggest that CNG4.3 forms a subunit of the native olfactory CNG channel. The expression of various CNG4 isoforms in retina and olfactory epithelium indicates that the CNG4 subunit may be necessary for normal function of both photoreceptor and olfactory CNG channels.

Developmental emergence of different forms of neuromodulation in Aplysia sensory neurons

The capacity for neuromodulation and biophysical plasticity is a defining feature of most mature neuronal cell types. In several cases, modulation at the level of the individual neuron has been causally linked to changes in the functional output of a neuronal circuit and subsequent adaptive changes in the organism’s behavioral responses. Understanding how such capacity for neuromodulation develops therefore may provide insights into the mechanisms both of neuronal development and learning and memory. We have examined the development of multiple forms of neuromodulation triggered by a common neurotransmitter, serotonin, in the pleural sensory neurons of Aplysia californica. We have found that multiple signaling cascades within a single neuron develop sequentially, with some being expressed only very late in development. In addition, our data suggest a model in which, within a single neuromodulatory pathway, the elements of the signaling cascade are developmentally expressed in a “retrograde” manner with the ionic channel that is modulated appearing early in development, functional elements in the second messenger cascade appearing later, and finally, coupling of the second messenger cascade to the serotonin receptor appearing quite late. These studies provide the characterization of the development of neuromodulation at the level of an identified cell type and offer insights into the potential roles of neuromodulatory processes in development and adult plasticity.

Nested expression domains for odorant receptors in zebrafish olfactory epithelium

The mapping of high-dimensional olfactory stimuli onto the two-dimensional surface of the nasal sensory epithelium constitutes the first step in the neuronal encoding of olfactory input. We have used zebrafish as a model system to analyze the spatial distribution of odorant receptor molecules in the olfactory epithelium by quantitative in situ hybridization. To this end, we have cloned 10 very divergent zebrafish odorant receptor molecules by PCR. Individual genes are expressed in sparse olfactory receptor neurons. Analysis of the position of labeled cells in a simplified coordinate system revealed three concentric, albeit overlapping, expression domains for the four odorant receptors analyzed in detail. Such regionalized expression should result in a corresponding segregation of functional response properties. This might represent the first step of spatial encoding of olfactory input or be essential for the development of the olfactory system.

Go protein-dependent survival of primary accessory olfactory neurons

Extensive G protein-coupled receptor families in both the main and accessory olfactory systems have been implicated in axonal targeting, sensory function, and cell survival. Although sensory function seems to be mediated by G proteins, axonal guidance and cell survival may be G protein-independent processes. In the accessory olfactory system, the Go-containing neurons in the basal vomeronasal organ (VNO) project to the posterior accessory olfactory bulb (AOB), whereas more apically located VNO neurons contain Gi2 and project to the anterior AOB. Herein, we investigate the organization of the accessory olfactory system in mice with a targeted deletion in the Goα gene. The accessory olfactory system seems normal at birth; however, postnatally, the number of Go-receptor-containing VNO neurons decreases by half, and apoptotic neurons are detected. The axons of VNO neurons remain restricted to the posterior AOB. The posterior AOB is reduced in size but contains a synaptophysin-positive layer with the normal number of glomeruli. The posterior AOB has reduced mitral cell c-Fos immunoreactivity, consistent with decreased sensory activation of Go protein-coupled VNO receptor neurons. Thus, in the accessory olfactory system, receptor-coupled G proteins are required for cell survival.

A dendrodendritic reciprocal synapse provides a recurrent excitatory connection in the olfactory bulb

Neuronal synchronization in the olfactory bulb has been proposed to arise from a diffuse action of glutamate released from mitral cells (MC, olfactory bulb relay neurons). According to this hypothesis, glutamate spills over from dendrodendritic synapses formed between MC and granule cells (GC, olfactory bulb interneurons) to activate neighboring MC. The excitation of MC is balanced by a strong inhibition from GC. Here we show that MC excitation is caused by glutamate released from bulbar interneurons located in the GC layer. These reciprocal synapses depend on an unusual, 2-amino-5-phosphonovaleric acid-resistant, N-methyl-d-aspartate receptor. This type of feedback excitation onto relay neurons may strengthen the original sensory input signal and further extend the function of the dendritic microcircuit within the main olfactory bulb.

Simple neural networks for the amplification and utilization of small changes in neuron firing rates

I describe physiologically plausible “voter-coincidence” neural networks such that secondary “coincidence” neurons fire on the simultaneous receipt of sufficiently large sets of input pulses from primary sets of neurons. The networks operate such that the firing rate of the secondary, output neurons increases (or decreases) sharply when the mean firing rate of primary neurons increases (or decreases) to a much smaller degree. In certain sensory systems, signals that are generally smaller than the noise levels of individual primary detectors, are manifest in very small increases in the firing rates of sets of afferent neurons. For such systems, this kind of network can act to generate relatively large changes in the firing rate of secondary “coincidence” neurons. These differential amplification systems can be cascaded to generate sharp, “yes–no” spike signals that can direct behavioral responses.

Genomic analysis of orthologous mouse and human olfactory receptor loci

Olfactory receptor (OR) genes represent ≈1% of genomic coding sequence in mammals, and these genes are clustered on multiple chromosomes in both the mouse and human genomes. We have taken a comparative genomics approach to identify features that may be involved in the dynamic evolution of this gene family and in the transcriptional control that results in a single OR gene expressed per olfactory neuron. We sequenced ≈350 kb of the murine P2 OR cluster and used synteny, gene linkage, and phylogenetic analysis to identify and sequence ≈111 kb of an orthologous cluster in the human genome. In total, 18 mouse and 8 human OR genes were identified, including 7 orthologs that appear to be functional in both species. Noncoding homology is evident between orthologs and generally is confined within the transcriptional unit. We find no evidence for common regulatory features shared among paralogs, and promoter regions generally do not contain strong promoter motifs. We discuss these observations, as well as OR clustering, in the context of evolutionary expansion and transcriptional regulation of OR repertoires.

Evidence for distinct signaling mechanisms in two mammalian olfactory sense organs.

In mammals, olfactory stimuli are detected by sensory neurons at two distinct sites: the olfactory epithelium (OE) of the nasal cavity and the neuroepithelium of the vomeronasal organ (VNO). While the OE can detect volatile chemicals released from numerous sources, the VNO appears to be specialized to detect pheromones that are emitted by other animals and that convey information of behavioral or physiological importance. The mechanisms underlying sensory transduction in the OE have been well studied and a number of components of the transduction cascade have been cloned. Here, we investigated sensory transduction in the VNO by asking whether VNO neurons express molecules that have been implicated in sensory transduction in the OE. Using in situ hybridization and Northern blot analyses, we found that most of the olfactory transduction components examined, including the guanine nucleotide binding protein alpha subunit (G-alpha-olf), adenylyl cyclase type III, and an olfactory cyclic nucleotide-gated (CNG) channel subunit (oCNC1), are not expressed by VNO sensory neurons. In contrast, VNO neurons do express a second olfactory CNG channel subunit (oCNC2). These results indicate that VNO sensory transduction is distinct from that in the OE but raise the possibility that, like OE sensory transduction, sensory transduction in the VNO might involve cyclic nucleotide-gated ion channels.

Single neuron control over a complex motor program.

While there are many instances of single neurons that can drive rhythmic stimulus-elicited motor programs, such neurons have seldom been found to be necessary for motor program function. In the isolated central nervous system of the marine mollusc Tritonia diomedea, brief stimulation (1 sec) of a peripheral nerve activates an interneuronal central pattern generator that produces the long-lasting (approximately 30-60 sec) motor program underlying the animal's rhythmic escape swim. Here, we identify a single interneuron, DRI (for dorsal ramp interneuron), that (i) conveys the sensory information from this stimulus to the swim central pattern generator, (ii) elicits the swim motor program when driven with intracellular stimulation, and (iii) blocks the depolarizing "ramp" input to the central pattern generator, and consequently the motor program itself, when hyperpolarized during the nerve stimulus. Because most of the sensory information appears to be funneled through this one neuron as it enters the pattern generator, DRI presents a striking example of single neuron control over a complex motor circuit.

Low doses of ivermectin cause sensory and locomotor disorders in dung beetles

Ivermectin is a veterinary pharmaceutical generally used to control the ecto- and endoparasites of livestock, but its use has resulted in adverse effects on coprophilous insects, causing population decline and biodiversity loss. There is currently no information regarding the direct effects of ivermectin on dung beetle physiology and behaviour. Here, based on electroantennography and spontaneous muscle force tests, we show sub-lethal disorders caused by ivermectin in sensory and locomotor systems of Scarabaeus cicatricosus, a key dung beetle species in Mediterranean ecosystems. Our findings show that ivermectin decreases the olfactory and locomotor capacity of dung beetles, preventing them from performing basic biological activities. These effects are observed at concentrations lower than those usually measured in the dung of treated livestock. Taking into account that ivermectin acts on both glutamate-gated and GABA-gated chloride ion channels of nerve and muscle cells, we predict that ivermectin’s effects at the physiological level could influence many members of the dung pat community. The results indicate that the decline of dung beetle populations could be related to the harmful effects of chemical contamination in the dung.

An ancient neurotrophin code in zebrafish; the Runx/Cbfβ complex specifies the fate switch from TrkB+ to TrkC+ nociceptors and sensory receptor expression

Thesis (Ph.D.)--University of Washington, 2016-06

Expression and putative role of lactoseries carbohydrates present on NCAM in the rat primary olfactory pathway

Primary olfactory neurons project axons from the olfactory neuroepithelium lining the nasal cavity to,the olfactory bulb in the brain. These axons grow within large mixed bundles in the olfactory nerve and then sort out into homotypic fascicles in the nerve fiber layer of the olfactory bulb before terminating in topographically fixed glomeruli. Carbohydrates expressed on the cell surface have been implicated in axon sorting within the nerve fiber layer. We have identified two novel subpopulations of primary olfactory neurons that express distinct alpha-extended lactoseries carbohydrates recognised by monoclonal antibodies LA4 and KH10. Both carbohydrate epitopes are present on novel glycoforms of the neural cell adhesion molecule, which we have named NOC-7 and NOC-8. Primary axon fasciculation is disrupted in vitro when interactions between these cell surface lactoseries carbohydrates and their endogenous binding molecules are inhibited by the LA4 and KH10 antibodies or lactosamine sugars. We report the expression of multiple members of the lactoseries binding galectin family in the primary olfactory system. In particular, galectin-3 is expressed by ensheathing cells surrounding nerve fascicles in the submucosa and nerve fiber layer, where it may mediate cross-linking of axons. Galectin-4, -7, and -8 are expressed by the primary olfactory axons as they grow from the nasal cavity to the olfactory bulb. A putative role for NOC-7 and NOC-8 in axon fasciculation and the expression of multiple galectins in the developing olfactory nerve suggest that these molecules may be involved in the formation of this pathway, particularly in the sorting of axons as they converge towards their target. (C) 2004Wiley-Liss, Inc.

Impaired memory and olfactory performance in NaSi-1 sulphate transporter deficient mice

In the present study, NaSi-l sulphate transporter knock-out (Nas1-/-) mice, an animal model of hyposulphataernia, were examined for spatial memory and learning in a Morris water maze, and for olfactory function in a cookie test. The Nas1-/- mice displayed significantly (P < 0.05) increased latencies to find an escape platform in the reversal teaming trials at 2 days but not 1 day after the last acquisition trial in a Morris water maze test. suggesting that Nas1-/- mice may have proactive memory interference. While the wild-type (Ncis1+/+) mice showed a significant (P < 0.02) decrease in time to locate a hidden food reward over four trials after overnight fasting, Nas1-/- mice did not change their performance, resulting in significantly (P < 0.05) higher latencies when compared to their Nas1+/+ littermates. There were no significant differences between Nas1-/- and Nas1+/+ mice in the cookie test after moderate food deprivation. In addition, both Nas1-/- and Nas1+/+ mice displayed similar escape latencies in the acquisition phase of the Morris water maze test, suggesting that learning, motivation, vision and motor skills required for the task may not be affected in Nas1-/- mice. This is the first study to demonstrate an impairment in memory and olfactory performance in the hyposulphataemic Nas1-/- mouse. (c) 2004 Elsevier B.V. All rights reserved.

Expression and role of complex carbohydrates in axon guidance in the olfactory system

Primary sensory neurons in the vertebrate olfactory systems are characterised by the differential expression of distinct cell surface carbohydrates. We show here that the histo-blood groups Sda (or CT1 antigen) and H are expressed by primary sensory neurons in the olfactory system, while the blood group A carbohydrate is expressed by a subset of vomeronasal neurons only in the developing accessory olfactory system. We have used both loss-of-function and gain-of-function approaches to manipulate expression of these carbohydrates in the olfactory system. In null mutant mice lacking the alpha(1,2)fucosyltransferase FUT1, the blood group H and A carbohydrates were not expressed in the olfactory systems which caused delayed development of the nerve fibre and glomerular layers in the main olfactory bulb. In contrast, ubiquitous expression of blood group A on olfactory axons in gain-of-function transgenic mice perturbed the ability of vomeronasal axons to terminate in the accessory olfactory bulb and affected the selective targeting of axons in the main olfactory bulb. During regeneration following bulbectomy, vomeronasal axons were unable to effectively sort out from the main olfactory axons when blood group A was misexpressed. These results provide in vivo evidence for a role of specific cell surface carbohydrates during development and regeneration of the olfactory nerve pathways.