Calcium imaging of adult olfactory epithelium reveals amines as important odor class in fish.

The odor space of aquatic organisms is by necessity quite different from that of air-breathing animals. The recognized odor classes in teleost fish include amino acids, bile acids, reproductive hormones, nucleotides, and a limited number of polyamines. Conversely, a significant portion of the fish olfactory receptor repertoire is composed of trace amine-associated receptors, generally assumed to be responsible for detecting amines. Zebrafish possess over one hundred of these receptors, but the responses of olfactory sensory neurons to amines have not been known so far. Here we examined odor responses of zebrafish olfactory epithelial explants at the cellular level, employing calcium imaging. We report that amines elicit strong responses in olfactory sensory neurons, with a time course characteristically different from that of ATP-responsive (basal) cells. A quantitative analysis of the laminar height distribution shows amine-responsive cells undistinguishable from ciliated neurons positive for olfactory marker protein. This distribution is significantly different from those measured for microvillous neurons positive for transient receptor potential channel 2 and basal cells positive for proliferating cell nuclear antigen. Our results suggest amines as an important odor class for teleost fish.

Zebrafish beta tubulin 1 expression is limited to the nervous system throughout development, and in the adult brain is restricted to a subset of proliferative regions.

Tubulin, the building block of microtubules, consists of an alpha and beta subunit, each in itself a family of several highly homologous isotypes. Abundance, tissue specificity, developmental regulation, and possibly function vary between isotypes. Six isotypes of beta tubulin (class I to class VI) have been cloned from several vertebrate species. Class I beta tubulin is believed to be widely expressed, but has not been studied by in situ hybridization in any vertebrate species so far. We have cloned a beta tubulin from zebrafish that appears most similar to other vertebrate class I tubulins and name it zbeta1 tubulin, accordingly. We report a distinct expression pattern of zbeta1 tubulin in the zebrafish embryo in restricted regions of the peripheral and central nervous system that comprise early-differentiating neurons. The expression pattern changes during development and in the adult zebrafish expression mostly is confined to a subset of proliferative zones that include the subependymal zone around the telencephalic ventricle, zones in the preoptic and hypothalamic area and in the olfactory epithelium. Thus, zbeta1 tubulin is expressed with remarkable selectivity during neuronal differentiation and neurogenesis in the embryonic and adult nervous system, respectively.

Selective targeting of zebrafish olfactory receptor neurons by the endogenous OMP promoter.

The olfactory nervous system of fish, in particular zebrafish, has become a valid model for that of higher vertebrates. However, no genetic markers for olfactory specific cell types, e.g. the olfactory receptor neurons, have been established in this species. Olfactory marker protein (OMP) is a reliable marker for olfactory receptor neurons in several other vertebrates. We have cloned zOMP, the zebrafish homologue of olfactory marker protein. During development, zOMP is expressed exclusively in the olfactory placode, presumably in olfactory receptor neurons, as shown by in situ hybridization. In the adult nasal epithelium zOMP is found restricted to the sensory region. zOMP appears to be a single gene, without close family members. The 5'-flanking region lacks most of the expected regulatory sequence motifs, both general and cell type-specific ones. Nevertheless, it drives reporter gene expression strongly and specifically in olfactory receptor neurons during the whole developmental period examined. Thus the zOMP promoter constitutes a powerful tool which should be useful to selectively introduce a wide variety of genetic modifications into olfactory receptor neurons.

An inexpensive mouse headholder suitable for optical recordings.

An inexpensive headholder for mice ranging from 3-week-old animals to adults was designed to provide reliable long-term head fixation. Its shape allows the direct access to the dorsal and ventral sides of the head which makes the headholder ideal for the use with both an upright and an inverted microscope. Because the headholder does not use a nose clamp, the apparatus allows surgery, stimulation of the olfactory system and concomitant optical recording of neuronal activity.

Odorant feature detection: activity mapping of structure response relationships in the zebrafish olfactory bulb.

The structural determinants of an odor molecule necessary and/or sufficient for interaction with the cognate olfactory receptor(s) are not known. Olfactory receptor neurons expressing the same olfactory receptor converge in the olfactory bulb. Thus, optical imaging of neuronal activity in the olfactory bulb can visualize at once the contributions by all the different olfactory receptors responsive to a particular odorant. We have used this technique to derive estimates about the structural requirements and minimal number of different zebrafish olfactory receptors that respond to a series of naturally occurring amino acids and some structurally related compounds. We report that the alpha-carboxyl group, the alpha-amino group, and l-conformation of the amino acid are all required for activation of amino acid-responsive receptors. Increasing carbon chain length recruits successively more receptors. With increasing concentrations, the activity patterns induced by a homolog series of amino acids became more similar to each other. At intermediate concentrations patterns were unique across substances and across concentrations. The introduction of a terminal amino group (charged) both recruits additional receptors and prevents binding to some of the receptors that were responsive to the unsubstituted analog. In contrast, the introduction of a beta-hydroxyl group (polar) excluded the odorants from some of the receptors that are capable of binding the unsubstituted analog. Cross-adaptation experiments independently confirmed these results. Thus, odorant detection requires several different receptors even for relatively simple odorants such as amino acids, and individual receptors require the presence of some molecular features, the absence of others, and tolerate still other molecular features.

Odor maps in the brain: spatial aspects of odor representation in sensory surface and olfactory bulb.

The olfactory sense detects and distinguishes a multitude of different odors. Recent progress in molecular as well as physiological approaches has elucidated basic principles of neuronal encoding of odorants, common to insects and vertebrates. The construction of neuronal representations for odors begins with the task of mapping the multidimensional odor space onto the two-dimensional sensory surface, and subsequently onto the olfactory bulb or antennal lobe. A distributed expression of odorant receptors, albeit restricted to subregions of the sensory surface (large, intermediate or small for zebrafish, mouse or drosophila, respectively), ensures a robust representation, insensitive to mechanical insult. Olfactory receptor neurons expressing the same odorant receptors converge to form a receptotopic map in the olfactory bulb or antennal lobe. The emerging coding principle is a chemotopic representation of odorants at the first brain level, realized either as combinatorial or as monospecific representation, depending on the odorant.

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.

Functional expression of odorant receptors of the zebrafish Danio rerio and of the nematode C. elegans in HEK293 cells.

Odorant receptors of zebrafish and C elegans were functionally expressed in vertebrate kidney cells (HEK293) using the eucaryotic expression vector pSMyc. Receptor-encoding cDNA cloned into this vector was expressed as a fusion protein with the N-terminal membrane import sequence of the guinea-pig serotonin receptor followed by a myc tag. Immunocytochemical evidence indicates that this strategy directs a protein with the predicted immunoreactivity and approximate molecular weight to the plasma membrane. Fish food extract (TetraMin) evoked a transient increase in intracellular [Ca2+] in HEK293 cells transiently transfected with plasmids containing cDNA for three fish odorant receptors and converted to stable cell lines. The effect of the extract was concentration dependent and limited to the fraction of the extract < 5 kDa. Pretreating the transfected cells with the PLC inhibitor U73122 reduced the odor-evoked signal. Fish food extract also evoked a transient increase in intracellular [Ca2+] in HEK293 cells transiently transfected with plasmids containing cDNA for single fish odorant receptors. Diacetyl evoked a transient increase in intracellular [Ca2+] in HEK293 cells transiently transfected with plasmids encoding the cDNA of ODR10, an odorant receptor of C. elegans suggested in other work to be specific for diacetyl. These results strongly imply that odorant receptors can be functionally expressed in HEK293 cells using this novel expression protocol.

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.

Olfaction in zebrafish: what does a tiny teleost tell us?

Zebrafish, Danio rerio, possess a well-developed sense of smell which governs a variety of behaviors. Both the number of odorant receptor genes and the number of modules in the olfactory bulb (glomeruli) are about an order of magnitude smaller than those of mammals. Nevertheless, the spatial organization of functional properties within the sensory surface and the olfactory bulb are comparable to those of mammals. The quantitatively reduced olfactory system of zebrafish, together with the suitability of this species for developmental and genetic studies, make zebrafish an interesting model system to study olfactory differentiation and neuronal representation of olfactory information.

Tissue-specific and ontogenetic regulation of LIF protein levels determined by quantitative enzyme immunoassay.

To define the physiological role of leukemia inhibitory factor (LIF), it is essential to localize sites of LIF synthesis in vivo. We generated polyclonal antibodies specific for native rat LIF, and developed a two-site immunoassay to detect 10 pg LIF/ml. Using this immunoassay, we determined LIF content of 18 organs, CNS regions, and ganglia throughout postnatal development of rats. High levels of LIF protein (1.0-11.0 ng/g tissue) are present in relatively few tissues: the uterus at late proestrus to estrus and on day 5 of pregnancy, ovary at estrus to early metestrus-1, footpads during early postnatal development and thymus throughout. Intermediate levels (0.5-1.0 ng) are detected in the gut, skin, skeletal muscle, pancreas and lung at one or more postnatal ages. Low levels (0.1-0.5 ng) are observed in most other non-nervous and nervous tissues. LIF protein levels do not completely correspond to reported LIF mRNA levels.

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.

Connectional topography in the zebrafish olfactory system: random positions but regular spacing of sensory neurons projecting to an individual glomerulus.

It is unknown how neuronal connections are specified in the olfactory system. To define rules of connectivity in this system, we investigated whether the projection of sensory neurons from the olfactory epithelium to the olfactory bulb is topographically ordered. By backtracking with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), we find that neurons projecting into a single identified glomerulus are widely dispersed over the olfactory epithelium. Their positions in the sensory surface do not predict their glomerulus specificity and are probably random. A statistical analysis reveals that neurons connected to the same glomerulus are spaced at distances of several cell diameters from each other. The convergence of projections to one point in the target area from neurons that are widely and evenly distributed in the sensory surface constitutes an unusual type of connectional topography that contrasts with the precise topological (neighborhood-preserving) maps found in other sensory systems. It may maximize the probability to detect odorants that activate a single glomerular unit.

Olfactory glomeruli in the zebrafish form an invariant pattern and are identifiable across animals.

Glomeruli are anatomical and possibly functional modules in the vertebrate olfactory bulb. We investigated the spatial arrangement of glomeruli in the olfactory bulbs of adult zebrafish (Brachydanio rerio). A solution of the lipophilic tracer Dil was injected into the nasal cavities. Axons of sensory neurons projecting from the olfactory epithelium into the bulb were traced anterogradely, thus labeling the whole population of glomeruli. The glomerular distribution was analyzed in detail by confocal laser-scanning microscopy. We find that a typical olfactory bulb contains a small number of about 80 glomeruli that have a stereotyped configuration in all animals investigated. All glomeruli exhibit bilateral symmetry. Twenty-two single glomeruli could be identified from animal to animal by their characteristic position and morphology. The remaining glomeruli either are embedded in glomerular plexus and therefore cannot be delineated reliably, or belong to a densely clustered subpopulation of on average 49 glomeruli in the dorsal olfactory bulb. No sexually dimorphic glomeruli were identified. To test whether glomerular constancy is specific for the zebrafish, we performed similar tracing experiments in the goldfish and found several indications for a similar invariance of glomeruli in this species. The remarkable stereotypy of this pattern is reminiscent of the insect olfactory system and has been demonstrated here for the first time in a vertebrate. It will now be possible to examine whether these identifiable glomeruli are functionally specialized in terms of odor processing. If so, zebrafish may emerge as a tractable model system for studies on olfactory coding.

The neurotrophic factor concept: a reexamination.

The neurotrophic factor concept in its basic form envisages that innervated tissues produce a signal for the innervating neurons for the selective limitation of neuronal death occurring during development (Purves, 1986; Oppenheim, 1991). This concept arose several decades ago on the basis of the observation that experimental manipulation of the amount of target tissue could modulate the size of neuronal populations. By making the survival of neurons dependent on their target, nature would provide a means to match neuron and target cell populations. NGF, discovered in the 1950s, represents the first known molecular realization of the neurotrophic factor concept. NGF was found to regulate survival, neurite growth, and neurotransmitter production of a particular neuronal type, the sympathetic neurons of the PNS. NGF produced by target cells is specifically bound and internalized by sympathetic neurons, followed by retrograde axonal transport of NGF to the cell soma, where NGF exerts its effects via the cotransported receptor molecule (Levi-Montalcini, 1987; Thoenen et al., 1987). Strictly speaking, increased neurite growth and neurotransmitter production are not trophic effects; however, I will use the term "neurotrophic" in the extended meaning of enhancing neuronal differentiation as well as neuronal survival. It was expected that these results could be generalized to a model of multiple, mutually independent, retrograde trophic messengers, which are synthesized in distinct target areas and act on restricted neuronal types (Fig. 1). This assumption leads to a conceptually simple way to arrange and maintain a variety of neuronal subsystems. One might call this a modular approach to the construction of the nervous system. The hypothesis of multiple retrograde signals has gained widespread experimental support in recent years. Originally proposed for the PNS, the model could be extended to the CNS, in which target neurons synthesize trophic factors for their afferent neurons (Ernfors et al., 1990b). In addition to NGF, a family of NGF-related molecules (now commonly called neurotrophins), which are thought to exert retrograde trophic influences (DiStefano et al., 1992), has been identified.

The cholinergic neuronal differentiation factor from heart cells is identical to leukemia inhibitory factor.

A protein secreted by cultured rat heart cells can direct the choice of neurotransmitter phenotype made by cultured rat sympathetic neurons. Structural analysis and biological assays demonstrated that this protein is identical to a protein that regulates the growth and differentiation of embryonic stem cells and myeloid cells, and that stimulates bone remodeling and acute-phase protein synthesis in hepatocytes. This protein has been termed D factor, DIA, DIF, DRF, HSFIII, and LIF. Thus, this cytokine, like IL-6 and TGF beta, regulates growth and differentiation in the embryo and in the adult in many tissues, now including the nervous system.

Nerve growth factor synthesis in cultured rat iris: modulation by endogenous transmitter substances.

Organ cultures of rat iris show a characteristic change in the levels of both nerve growth factor (NGF) and its mRNA: a rapid but transient initial increase is followed by a smaller but persistently elevated NGF synthesis. This time course may be influenced by release of a factor(s) from degenerating nerve terminals and/or by the lack of some factor(s) repressing NGF synthesis in vivo. We therefore analyzed the influence of biogenic amine transmitter substances and putative neuropeptides on this elevation of NGF synthesis in cultured iris. The marked increase of NGF synthesis seen initially in culture was not completely mimicked by any of the substances tested. A specific increase in NGF production up to 150% of control was observed only with cGMP. We also obtained some evidence that reaction to trauma following the culture procedure could enhance NGF production: cutting of irides into small pieces increased NGF production in culture up to 250% of control and, vice versa, treatment with 1 microM dexamethasone decreased NGF production to about 60% of control. However, the sympathetic neurotransmitter norepinephrine (NE) decreased both NGF and its mRNA levels specifically in a dose-dependent manner (0.01-1 mM) to a minimum of about 25% of control. In situ hybridization with mRNA(NGF)-specific probes showed that in cultures of dissociated iris cells all cells were capable of expressing mRNA(NGF), but that 0.1 mM NE preferentially decreased expression of mRNA(NGF) in smooth muscle cells. Thus, our results indicate that the sympathetic transmitter NE is capable of downregulating NGF synthesis in the target cells of sympathetic neurons.

Antibodies against mouse nerve growth factor interfere in vivo with the development of avian sensory and sympathetic neurones.

The monoclonal antibody 27/21 directed against mouse nerve growth factor (NGF) interferes in vivo with the survival of sensory dorsal root ganglion (DRG) neurones during the development of the quail embryo: the number of DRG neurones at embryonic day 11 (E11) was reduced by about 30% in embryos treated with the antibody between E3 and E11. Neurone numbers in the nodose ganglion were not affected. The effect of NGF antibodies on sympathetic neurones was assessed by determining the levels of the adrenergic marker enzyme tyrosine hydroxylase. Both total tyrosine hydroxylase activity and protein levels in sympathetic chains were reduced by about 30% in embryos treated with 27/21 antibody but not in embryos treated with a control antibody. The 27/21 antibody cross-reacts with chick NGF-like activity as shown in vitro by the ability of the antibody to partially block the survival activity of chick-embryo-fibroblast-conditioned medium for E9 chick DRG neurones.

Developmental changes of nerve growth factor levels in sympathetic ganglia and their target organs.

The predominant source of nerve growth factor (NGF) used by mature sympathetic neurons originates in their target organs (Heumann, R., Korsching, S., Scott, J., and Thoenen, H. (1984), EMBO J. 3, 3183-3189; Korsching, S., and Thoenen, H. (1985), J. Neurosci. 5, 1058-1061). We have determined the NGF content of two sympathetically innervated mouse organs, submandibular gland and heart ventricle, and of sympathetic ganglia from mouse and rat between embryonic Day 12 (E12) and adulthood. NGF levels were measured by a two-site enzyme immunassay (Korsching, S., and Thoenen, H. (1983), Proc. Natl. Acad. Sci. USA 80, 3513-3516). In heart ventricle and submandibular gland, NGF first became detectable around the time of initial innervation by sympathetic neurons (E12 and E13, respectively) and increased respectively 14- and 7-fold in the following 2 days, to reach adult levels already at E14 for heart ventricle (1.4 +/- 0.2 ng NGF/g wet wt). NGF in the superior cervical ganglion (SCG) was first detected at the same time as in its target organ, the submandibular gland. NGF content in the SCG then increased 6-fold during the next 2 days and continued to increase until the end of the third postnatal week, when adult levels were reached. Although the levels of NGF in the adult mouse submandibular gland are sexually dimorphic and six orders of magnitude higher than those in other sympathetic target organs, no sex difference in the NGF content was found in either developing submandibular gland or SCG until the end of the third postnatal week. Moreover, the steep NGF increase observed in the male submandibular gland after postnatal Day 18 (250-fold within the following 3 days and up to the 55,000-fold in the next 7 days) was not reflected in a corresponding increase in the NGF content of the male SCG. These data indicate that, in accordance with earlier findings (see Levi-Montalcini, R., and Angeletti, P. U. (1968), Physiol. Rev. 48, 534-569), SCG neurons do not have access to the large amounts of NGF synthesized during and after adolescence in the mouse submandibular gland. Our results support the concept that initial fiber outgrowth of sympathetic neurons is neither dependent on NGF nor mediated by it. The time course of NGF levels in the SCG is consistent with the concept that sympathetic neurons are provided with NGF by means of retrograde axonal transport from the innervated organs already early in development.