Search

In the olfactory bulb (OB) of zebrafish and other species, odors evoke fast oscillatory population activity and specific firing rate patterns across mitral cells (MCs). This activity evolves over a few hundred milliseconds from the onset of the odor stimulus. Action potentials of odor-specific MC subsets phase-lock to the oscillation, defining small and distributed ensembles within the MC population output. We found that oscillatory field potentials in the zebrafish OB propagate across the OB in waves. Phase-locked MC action potentials, however, were synchronized without a time lag. Firing rate patterns across MCs analyzed with low temporal resolution were informative about odor identity. When the sensitivity for phase-locked spiking was increased, activity patterns became progressively more informative about odor category. Hence, information about complementary stimulus features is conveyed simultaneously by the same population of neurons and can be retrieved selectively by biologically plausible mechanisms, indicating that seemingly alternative coding strategies operating on different time scales may coexist.

Glomeruli in the olfactory bulb are anatomically discrete modules receiving input from idiotypic olfactory sensory neurons. To examine the functional organization of sensory inputs to individual glomeruli, we loaded olfactory sensory neurons with a Ca 2+ indicator and measured odorant-evoked presynaptic Ca 2+ signals within single glomeruli by using two-photon microscopy in anaesthetized mice. Odorants evoked patterns of discrete Ca 2+ signals throughout the neuropil of a glomerulus. Across glomeruli, Ca 2+ signals occurred with equal probability in all glomerular regions. Within single glomeruli, the pattern of intraglomerular Ca 2+ signals was indistinguishable for stimuli of different duration, identity, and concentration. Moreover, the response time course of the signals was similar throughout the glomerulus. Hence, sensory inputs to individual glomeruli are spatially heterogeneous but seem to be functionally indiscriminate. These results support the view of olfactory glomeruli as functional units in representing sensory information.

L-type Ca2+channels (LTCCs) drive the bulk of voltage-gated Ca2+entry in vertebrate inner ear hair cells (HCs) and are essential for mammalian auditory processing. LTCC currents have been implicated in neurotransmitter release at the HC afferent active zone, the ribbon synapse. It is likely that LTCCs play a direct role in vesicle fusion; however, the subcellular localization of the channels in HCs has not been fully resolved. Via positional cloning, we show that mutations in a zebrafish LTCC encoding gene,cav1.3a, underlie the auditory-vestibular defects ofgemini(gem) circler mutants.gemhomozygous receptor mutant HCs display normal cell viability, afferent synaptogenesis, and peripheral innervation, yet exhibit strongly reduced extracellular potentials (∼50% of wild-type potentials). Apical FM1-43 uptake, however, is unaffected ingemmutant HCs, suggesting that mechanotransduction channels are functional. Using a Gem-specific antibody, we show that the bulk of Gem/Cav1.3a immunoreactivity in HCs is restricted to basally located focal spots. The number and location of focal spots relative to nerve terminals, and their remarkable ring-shaped structure, which is reminiscent of synaptic dense bodies, are consistent with Gem/Cav1.3a channels clustering at HC ribbon synapses.

Many bacterial pathogens secrete proteins that activate host trypsinogen-like enzyme precursors, most notably the proenzymes of the blood coagulation and fibrinolysis systems. Staphylococcus aureus, an important human pathogen implicated in sepsis and endocarditis, secretes the cofactor staphylocoagulase, which activates prothrombin, without the usual proteolytic cleavages, to directly initiate blood clotting. Here we present the 2.2 A crystal structures of human alpha-thrombin and prethrombin-2 bound to a fully active staphylocoagulase variant. The cofactor consists of two domains, each with three-helix bundles; this is a novel fold that is distinct from known serine proteinase activators, particularly the streptococcal plasminogen activator streptokinase. The staphylocoagulase fold is conserved in other bacterial plasma-protein-binding factors and extracellular-matrix-binding factors. Kinetic studies confirm the importance of isoleucine 1 and valine 2 at the amino terminus of staphylocoagulase for zymogen activation. In addition to making contacts with the 148 loop and (pro)exosite I of prethrombin-2, staphylocoagulase inserts its N-terminal peptide into the activation pocket of bound prethrombin-2, allosterically inducing functional catalytic machinery. These investigations demonstrate unambiguously the validity of the zymogen-activation mechanism known as 'molecular sexuality'.

Neuropeptides play important roles in chemical signalling in the central and peripheral nervous systems. One of the largest families of neuropeptides is that of the FMRFamide-like peptides (FLPs). This paper reviews what is known about the physiological functions of FLPs in crustaceans, focussing on the cardiovascular, digestive and neuromuscular systems.

The zebrafish has various advantages as a model organism to analyze the structure and function of neural circuits but efficient viruses or other tools for fast gene transfer are lacking. We show that transgenes can be introduced directly into the adult zebrafish brain by herpes simplex type I viruses (HSV-1) or electroporation. We developed a new procedure to target electroporation to defined brain areas and identified promoters that produced strong long-term expression. The fast workflow of electroporation was exploited to express multiple channelrhodopsin-2 variants and genetically encoded calcium indicators in telencephalic neurons for measurements of neuronal activity and synaptic connectivity. The results demonstrate that HSV-1 and targeted electroporation are efficient tools for gene delivery into the zebrafish brain, similar to adeno-associated viruses and lentiviruses in other species. These methods fill an important gap in the spectrum of molecular tools for zebrafish and are likely to have a wide range of applications.

Odors are first detected by olfactory sensory neurons (OSNs) and evoke stimulus-specific patterns of activation across the input channels of the olfactory bulb (OB), the glomeruli. The output of the OB consists of spatiotemporal activity patterns across mitral/tufted cells that are conveyed to multiple pallial and subpallial target areas. In the main olfactory system of vertebrates, as well as in the olfactory system of insects, odor information is encoded by distributed patterns of activity across a large number of glomeruli or neurons. Ca2+ imaging has therefore become an important approach used to analyse the encoding and processing of olfactory information by populations of glomeruli or neurons. Experiments in the intact olfactory system are important to maintain the integrity of the system, to analyse activity patterns evoked by natural odors, and to examine the influence of active sampling strategies, such as sniffing in mammals. This protocol focuses on how to visualize glomerular Ca2+ signals after loading a dextran-coupled Ca2+ indicator into OSNs. Separate procedures are described for zebrafish and mice.

Olfactory imprinting on environmental, population- and kin-specific cues is a specific form of life-long memory promoting homing of salmon to their natal rivers and the return of coral reef fish to natal sites. Despite its ecological significance, natural chemicals for olfactory imprinting have not been identified yet. Here, we show that MHC peptides function as chemical signals for olfactory imprinting in zebrafish. We found that MHC peptides consisting of nine amino acids elicit olfactory imprinting and subsequent kin recognition depending on the MHC genotype of the fish. In vivo calcium imaging shows that some olfactory bulb neurons are highly sensitive to MHC peptides with a detection threshold at 1 pM or lower, indicating that MHC peptides are potent olfactory stimuli. Responses to MHC peptides overlapped spatially with responses to kin odour but not food odour, consistent with the hypothesis that MHC peptides are natural signals for olfactory imprinting.

Recent studies of the olfactory bulb (vertebrates) and antennal lobe (insects) have elucidated how odor information is represented by the identity, synchronization, slow temporal dynamics and position of active neurons in an ensemble. Odor representations are dynamically reorganized both during the course of a stimulus and with experience. These results provide new insights into the logic of odor representations and dynamical network computations.

beta-Selenolo[3,2-b]pyrrolyl-L-alanine that mimics tryptophan with the benzene ring of the indole moiety replaced by selenophene, was incorporated into human annexin V and barstar. This was achieved by fermentation and expression in a Trp-auxotrophic Escherichia coli host strain using the selective pressure incorporation method. The seleno- proteins were obtained in yields comparable to those of the wild-type proteins and exhibit full crystallographic isomorphism to the parent proteins, but expectedly show altered absorbance profiles and quenched tryptophan fluorescence. Since the occurrence of tryptophan residues in proteins is rare, incorporation of the electron-rich selenium-containing tryptophan surrogate into proteins represents a useful supplementation and even a promising novel alternative to selenomethionine for solving the phase problem in protein X-ray crystallography.

The discovery that certain neurons' odour responses differ between individual fruit flies, but are consistent across the hemispheres of each fly's brain, indicates that sensory processing depends on an individual's experience. See Letter p.258 Neuronal representations of sensory stimuli tend to become sparse and decorrelated — with, say, different odours giving rise to fewer neuronal spikes in rare neurons — as signal processing moves up to higher brain layers. Based on comprehensive recording from the fourth neuronal layer in Drosophila's olfactory processing centre — the mushroom body output neurons (MBONs) — Glenn Turner and colleagues find instead some highly correlated tuning curves. They go on to show that such reformatting of sensory representations, from highly stimulus-specific information in sparse layers converging onto a limited number of final motor outputs, implicates neuronal plasticity and thus varies flexibly from animal to animal.

The molecular basis of sensory hair cell mechanotransduction is largely unknown. In order to identify genes that are essential for mechanosensory hair cell function, we characterized a group of recently isolated zebrafish motility mutants. These mutants are defective in balance and swim in circles but have no obvious morphological defects. We examined the mutants using calcium imaging of acoustic-vibrational and tactile escape responses, high resolution microscopy of sensory neuroepithelia in live larvae, and recordings of extracellular hair cell potentials (microphonics). Based on the analyses, we have identified several classes of genes. Mutations in sputnik and mariner affect hair bundle integrity. Mutant astronaut and cosmonaut hair cells have relatively normal microphonics and thus appear to affect events downstream of mechanotransduction. Mutant orbiter, mercury, and gemini larvae have normal hair cell morphology and yet do not respond to acoustic-vibrational stimuli. The microphonics of lateral line hair cells of orbiter, mercury, and gemini larvae are absent or strongly reduced. Therefore, these genes may encode components of the transduction apparatus.

Friedrich, Rainer W., G. F. Molnar, Michael Schiebe, and A. Joffre Mercier. Protein kinase C is required for long-lasting synaptic enhancement by the neuropeptide DRNFLRFamide in crayfish. J. Neurophysiol. 79: 1127–1131, 1998. The FMRFamide-related neuropeptide AspArgAsnPheLeuArgPhe-NH2(DRNFLRFamide, DF2) induces a long-lasting enhancement of synaptic transmission at neuromuscular junctions on the crayfish deep abdominal extensor muscles. Here we investigated the function of protein kinase C (PKC) in this effect because PKC has been implied in the control of long-term synaptic modulation in other systems. The general kinase antagonist staurosporine reduced both the initial increase in excitatory postsynaptic potential (EPSP) amplitude and the duration of synaptic enhancement. Unlike staurosporine, the selective PKC inhibitors, chelerythrine and bisindolylmaleimide, augmented the initial EPSP increase. However, like staurosporine, they also reduced the duration of synaptic enhancement. The PKC activator, phorbol-12-myristate 13-acetate, induced a long-lasting synaptic enhancement that was blocked by chelerythrine. These results show that synaptic enhancement by DF2is mediated by different intracellular signaling systems that act in temporal sequence. The initial increase in EPSP amplitudes is negatively regulated by PKC and involves another, staurosporine-sensitive, kinase; whereas, the maintenance of synaptic enhancement requires PKC.

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.

Olfactory imprinting on environmental, population- and kin-specific cues is a specific form of life-long memory promoting homing of salmon to their natal rivers and the return of coral reef fish to natal sites. Despite its ecological significance, natural chemicals for olfactory imprinting have not been identified yet. Here, we show that MHC peptides function as chemical signals for olfactory imprinting in zebrafish. We found that MHC peptides consisting of nine amino acids elicit olfactory imprinting and subsequent kin recognition depending on the MHC genotype of the fish. In vivo calcium imaging shows that some olfactory bulb neurons are highly sensitive to MHC peptides with a detection threshold at 1 pM or lower, indicating that MHC peptides are potent olfactory stimuli. Responses to MHC peptides overlapped spatially with responses to kin odour but not food odour, consistent with the hypothesis that MHC peptides are natural signals for olfactory imprinting.

Robustness of neuronal activity patterns against variations in input intensity is critical for neuronal computations. We found that odor representations in the olfactory bulb were stabilized by interneurons that were densely coupled to the output neurons by electrical and GABAergic synapses. This interneuron network modulated responses of output neurons as a function of stimulus intensity in two ways: it globally boosted responses to weak odors, but attenuated responses to strong odors, and it increased the sensitivity of some output neurons, but decreased the sensitivity of others. These effects are closely related to strategies used in engineering to increase dynamic range. Together, they maintained not only the mean, but also the distribution, of activity across the population of output neurons within narrow limits, which is important for pattern classification. Neuronal circuits in the olfactory bulb therefore stabilize combinatorial sensory representations against variations in stimulus intensity by generic mechanisms.

The main olfactory system encodes information about molecules in a combinatorial fashion by distributed spatiotemporal activity patterns. As activity propagates from sensory neurons to the olfactory bulb and to higher brain areas, odor information is processed by multiple transformations of these activity patterns. This review discusses neuronal computations associated with such transformations in the olfactory system of zebrafish, a small vertebrate that offers advantages for the quantitative analysis and manipulation of neuronal activity in the intact brain. The review focuses on pattern decorrelation in the olfactory bulb and on the readout of multiplexed sensory representations in the telencephalic area Dp, the homolog of the olfactory cortex. These computations are difficult to study in larger species and may provide insights into general information-processing strategies in the brain.

The clever choice of animal models has been instrumental for many breakthrough discoveries in life sciences. One of the outstanding challenges in neuroscience is the in-depth analysis of neuronal circuits to understand how interactions between large numbers of neurons give rise to the computational power of the brain. A promising model organism to address this challenge is the zebrafish, not only because it is cheap, transparent and accessible to sophisticated genetic manipulations but also because it offers unique advantages for quantitative analyses of circuit structure and function. One of the most important advantages of zebrafish is its small brain size, both at larval and adult stages. Small brains enable exhaustive measurements of neuronal activity patterns by optical imaging and facilitate large-scale reconstructions of wiring diagrams by electron microscopic approaches. Such information is important, and probably essential, to obtain mechanistic insights into neuronal computations underlying higher brain functions and dysfunctions. This review provides a brief overview over current methods and motivations for dense reconstructions of neuronal activity and connectivity patterns. It then discusses selective advantages of zebrafish and provides examples how these advantages are exploited to study neuronal computations in the olfactory bulb.

Complex motor behaviors are thought to be coordinated by networks of brain nuclei that may control different elementary motor programs. Transparent zebrafish larvae offer the opportunity to analyze the functional organization of motor control networks by optical manipulations of neuronal activity during behavior. We examined motor behavior in transgenic larvae expressing channelrhodopsin-2 throughout many neurons in the brain. Wide-field optical stimulation triggered backward and rotating movements caused by the repeated execution of J-turns, a specific motor program that normally occurs during prey capture. Although optically-evoked activity was widespread, behavioral responses were highly coordinated and lateralized. 3-D mapping of behavioral responses to local optical stimuli revealed that J-turns can be triggered specifically in the anterior-ventral optic tectum (avOT) and/or the adjacent pretectum. These results suggest that the execution of J-turns is controlled by a small group of neurons in the midbrain that may act as a command center. The identification of a brain area controlling a defined motor program involved in prey capture is a step toward a comprehensive analysis of neuronal circuits mediating sensorimotor behaviors of zebrafish.

The olfactory system encodes information about molecules by spatiotemporal patterns of activity across distributed populations of neurons and extracts information from these patterns to control specific behaviors. Recent studies usedin vivorecordings, optogenetics, and other methods to analyze the mechanisms by which odor information is encoded and processed in the olfactory system, the functional connectivity within and between olfactory brain areas, and the impact of spatiotemporal patterning of neuronal activity on higher-order neurons and behavioral outputs. The results give rise to a faceted picture of olfactory processing and provide insights into fundamental mechanisms underlying neuronal computations. This review focuses on some of this work presented in a Mini-Symposium at the Annual Meeting of the Society for Neuroscience in 2012.

The olfactory system has become a popular model to study the function of neuronal circuits and the molecular and cellular mechanisms underlying the development of neurons and their connections. An excellent model to combine studies of function and development is the zebrafish because it not only permits sophisticated molecular and genetic analyses of development, but also functional measurements of neuronal activity patterns in the intact brain. This article reviews insights into the functional development of the olfactory system that have been obtained in zebrafish. The focus is on the specification of olfactory sensory neurons (OSNs), the mechanisms controlling odorant receptor expression and OSN identity, the pathfinding of OSN axons towards target glomeruli in the olfactory bulb (OB), the development of glomeruli and functional topographic maps in the OB, and the development of inhibitory interneurons in the OB.

Dopamine (DA) is an important modulator of synaptic transmission and plasticity that is causally involved in fundamental brain functions and dysfunctions. We examined the dopaminergic modulation of synaptic transmission and sensory responses in telencephalic area Dp of zebrafish, the homologue of olfactory cortex. By combining anatomical tracing and immunohistochemistry, we detected no DA neurons in Dp itself but long-range dopaminergic input from multiple other brain areas. Whole-cell recordings revealed no obvious effects of DA on membrane potential or input resistance in the majority of Dp neurons. Electrical stimulation of the olfactory tracts produced a complex sequence of synaptic currents in Dp neurons. DA selectively decreased inhibitory currents with little or no effect on excitatory components. Multiphoton calcium imaging showed that population responses of Dp neurons to olfactory tract stimulation or odor application were enhanced by DA, consistent with its effect on inhibitory synaptic transmission. These effects of DA were blocked by an antagonist of D2-like receptors. DA therefore disinhibits and reorganizes sensory responses in Dp. This modulation may affect sensory perception and could be involved in the experience-dependent modification of odor representations.

In order to analyze neuronal computations in the first olfactory processing center, the olfactory bulb, we measured odor-evoked activity patterns across large numbers of neurons within the intact olfactory bulb of zebrafish using optical and electrophysiological methods. We found that the olfactory bulb performs multiple computations including a decorrelation of overlapping inputs, a multiplexing of complementary information, and gain control. Patterns of olfactory bulb output activity are reorganized during the initial phase of an odor response, resulting in a partial loss of the topographic representation of molecular features and in the decorrelation of activity patterns evoked by similar stimuli. Physiological, pharmacological, and computational results provide initial insights into the mechanisms underlying these computations.

gamma-Aminobutyric acid (GABA)ergic synapses are thought to play pivotal roles in the processing of activity patterns in the olfactory bulb (OB), but their functions have been difficult to study during odor responses in the intact system. We pharmacologically manipulated GABA(A) and GABA(B) receptors in the OB of zebrafish and analysed the effects on odor responses of the output neurons, the mitral cells (MCs), by electrophysiological recordings and temporally deconvolved two-photon Ca2+ imaging. The blockade of GABA(B) receptors enhanced presynaptic Ca2+ influx into afferent axon terminals, and changed the amplitude and time course of a subset of MC responses, indicating that GABA(B) receptors have a modulatory influence on OB output activity. The blockade of GABA(A) receptors induced epileptiform firing, enhanced excitatory responses and abolished fast oscillations in the local field potential. Moreover, the topological reorganization and decorrelation of MC activity patterns during the initial phase of the response was perturbed. These results indicate that GABA(A) receptor-containing circuits participate in the balance of excitation and inhibition, the regulation of total OB output activity, the synchronization of odor-dependent neuronal ensembles, and the reorganization of odor-encoding activity patterns. GABA(A) and GABA(B) receptors are therefore differentially involved in multiple functions of neuronal circuits in the OB.

Odors are initially represented in the olfactory bulb (OB) by patterns of sensory input across the array of glomeruli. Although activated glomeruli are often widely distributed, glomeruli responding to stimuli sharing molecular features tend to be loosely clustered and thus establish a fractured chemotopic map. Neuronal circuits in the OB transform glomerular patterns of sensory input into spatiotemporal patterns of output activity and thereby extract information about a stimulus. It is, however, unknown whether the chemotopic spatial organization of glomerular inputs is maintained during these computations. To explore this issue, we measured spatiotemporal patterns of odor-evoked activity across thousands of individual neurons in the zebrafish OB by temporally deconvolved two-photon Ca2+ imaging. Mitral cells and interneurons were distinguished by transgenic markers and exhibited different response selectivities. Shortly after response onset, activity patterns exhibited foci of activity associated with certain chemical features throughout all layers. During the subsequent few hundred milliseconds, however, MC activity was locally sparsened within the initial foci in an odor-specific manner. As a consequence, chemotopic maps disappeared and activity patterns became more informative about precise odor identity. Hence, chemotopic maps of glomerular input activity are initially transmitted to OB outputs, but not maintained during pattern processing. Nevertheless, transient chemotopic maps may support neuronal computations by establishing important synaptic interactions within the circuit. These results provide insights into the functional topology of neural activity patterns and its potential role in circuit function., Author Summary Many sensory brain areas contain topographic maps where the physical location of neuronal activity contains information about a stimulus feature. In the first central processing center of the olfactory pathway, the olfactory bulb, chemically distinct odors often elicit spatially segregated input activity so that general chemical features are initially represented in a topographic fashion. It is, however, unclear whether this “chemotopic” organization of odor representations is maintained at subsequent stages of odor processing. To address this question, we visualized activity patterns across thousands of individual neurons in the intact olfactory bulb of zebrafish over time using two-photon calcium imaging. Our results demonstrate that odor-evoked activity across the output neurons of the olfactory bulb is chemotopically organized shortly after stimulus onset but becomes more widely distributed during the subsequent few hundred milliseconds of the response. This reorganization of olfactory bulb output activity is most likely mediated by inhibitory feedback and reduces the redundancy in activity patterns evoked by related stimuli. These results indicate that topographically organized activity maps in the olfactory bulb are not maintained during information processing, but contribute to the function of local circuits., Two-photon calcium imaging in the zebrafish olfactory bulb reveals that mitral cells show more selective responses to odors than interneurons, and odor-evoked firing patterns of populations of mitral cells evolve over hundreds of milliseconds to become more distinct for different odors, thus providing more information about odor identity.

Human tissue kallikrein 4 (hK4) belongs to a 15-member family of closely related serine proteinases. hK4 is predominantly expressed in prostate, activates hK3/PSA, and is up-regulated in prostate and ovarian cancer. We have identified active monomers of recombinant hK4 besides inactive oligomers in solution. hK4 crystallised in the presence of zinc, nickel, and cobalt ions in three crystal forms containing cyclic tetramers and octamers. These structures display a novel metal site between His25 and Glu77 that links the 70-80 loop with the N-terminal segment. Micromolar zinc as present in prostatic fluid inhibits the enzymatic activity of hK4 against fluorogenic substrates. In our measurements, wild-type hK4 exhibited a zinc inhibition constant (IC50) of 16 microM including a permanent residual activity, in contrast to the zinc-independent mutants H25A and E77A. Since the Ile16 N terminus of wild-type hK4 becomes more accessible for acetylating agents in the presence of zinc, we propose that zinc affects the hK4 active site via the salt-bridge formed between the N terminus and Asp194 required for a functional active site. hK4 possesses an unusual 99-loop that creates a groove-like acidic S2 subsite. These findings explain the observed specificity of hK4 for the P1 to P4 substrate residues. Moreover, hK4 shows a negatively charged surface patch, which may represent an exosite for prime-side substrate recognition.

Odorants are first represented in the brain by distributed patterns of activity in the olfactory bulb (OB). Although neurons downstream of sensory inputs respond to odorants with temporally structured activity, sensory inputs to glomeruli are typically described as static maps. Here, we imaged the temporal dynamics of receptor neuron input to the OB with a calcium-sensitive dye in the olfactory receptor nerve terminals in anesthetized mice. We found that diverse, glomerulus- and odorant-dependent temporal dynamics are present even at this initial input stage. Instantaneous spatial patterns of receptor input to glomeruli changed both within and between respiration cycles. Glomerular odorant responses differed in amplitude, latency, rise time, and degree of modulation by sniffing in an odorant-specific manner. Pattern dynamics within the first respiration cycle recurred in a similar manner during consecutive cycles. When sniff rate was increased artificially, pattern dynamics were preserved in the first sniff but were attenuated during subsequent sniffs. Temporal response properties were consistent across individuals on a coarse regional scale and on a fine scale of individual glomeruli. Latency and magnitude of glomerular inputs were only weakly correlated and might therefore convey independent odorant information. These data demonstrate that glomerular maps of primary sensory input to the OB are temporally dynamic. These dynamics may contribute to the representation of odorant information and affect information processing in the central olfactory system of rodents.

Understanding how complex neuronal circuits in the brain perform advanced computations is a central question in neuroscience that can only be addressed using a combination of approaches, including neurophysiology and behavioral analyses. In the olfactory bulb, neurophysiological studies have revealed that neuronal interactions reorganize odor-evoked activity patterns so that their discriminability is enhanced. Recent behavioral studies have examined the role of this computation in odor discrimination tasks and generated working models of behavioral odor discrimination strategies. The results appear consistent with a role of pattern reorganization in odor discrimination behavior but further studies are necessary to resolve this issue. These studies advance the understanding of neuronal circuit function in the olfactory bulb and illustrate benefits and caveats of comparing behavioral and neurophysiological results.

Methods to record action potential (AP) firing in many individual neurons are essential to unravel the function of complex neuronal circuits in the brain. A promising approach is bolus loading of Ca(2+) indicators combined with multiphoton microscopy. Currently, however, this technique lacks cell-type specificity, has low temporal resolution and cannot resolve complex temporal firing patterns. Here we present simple solutions to these problems. We identified neuron types by colocalizing Ca(2+) signals of a red-fluorescing indicator with genetically encoded markers. We reconstructed firing rate changes from Ca(2+) signals by temporal deconvolution. This technique is efficient, dramatically enhances temporal resolution, facilitates data interpretation and permits analysis of odor-response patterns across thousands of neurons in the zebrafish olfactory bulb. Hence, temporally deconvolved Ca(2+) imaging (TDCa imaging) resolves limitations of current optical recording techniques and is likely to be widely applicable because of its simplicity, robustness and generic principle.

Here, we present a series of thrombin inhibitors that were generated by using powerful computer-assisted multiparameter optimization process. The process was organized in design cycles, starting with a set of randomly chosen molecules. Each cycle combined combinatorial synthesis, multiparameter characterization of compounds in a variety of bioassays, and algorithmic processing of the data to devise a set of compounds to be synthesized in the next cycle. The identified lead compounds exhibited thrombin inhibitory constants in the lower nanomolar range. They are by far the most selective synthetic thrombin inhibitors, with selectivities of >100,000-fold toward other proteases such as Factor Xa, Factor XIIa, urokinase, plasmin, and Plasma kallikrein. Furthermore, these compounds exhibit a favorable profile, comprising nontoxicity, high metabolic stability, low serum protein binding, good solubility, high anticoagulant activity, and a slow and exclusively renal elimination from the circulation in a rat model. Finally, x-ray crystallographic analysis of a thrombin–inhibitor complex revealed a binding mode with a neutral moiety in the S1 pocket of thrombin.

A small molecule called optovin acts as a light-sensitive ligand for an ion channel involved in the detection of painful sensory stimuli. This unexpected finding emerged from a behavioral screen in zebrafish and provides a tool for the remote control of pain by light.

Components of odor mixtures often are not perceived individually, suggesting that neural representations of mixtures are not simple combinations of the representations of the components. We studied odor responses to binary mixtures of amino acids and food extracts at different processing stages in the olfactory bulb (OB) of zebrafish. Odor-evoked input to the OB was measured by imaging Ca2+signals in afferents to olfactory glomeruli. Activity patterns evoked by mixtures were predictable within narrow limits from the component patterns, indicating that mixture interactions in the peripheral olfactory system are weak. OB output neurons, the mitral cells (MCs), were recorded extra- and intracellularly and responded to odors with stimulus-dependent temporal firing rate modulations. Responses to mixtures of amino acids often were dominated by one of the component responses. Responses to mixtures of food extracts, in contrast, were more distinct from both component responses. These results show that mixture interactions can result from processing in the OB. Moreover, our data indicate that mixture interactions in the OB become more pronounced with increasing overlap of input activity patterns evoked by the components. Emerging from these results are rules of mixture interactions that may explain behavioral data and provide a basis for understanding the processing of natural odor stimuli in the OB.

In the olfactory system, odors are represented by spatially-organized patterns of neural activity in the olfactory bulb, the first synaptic level of the olfactory pathway. We have visualized these patterns by loading mouse olfactory receptor neurons with calcium-sensitive dye and imaging odorant-evoked fluorescence signals from their axon terminals. This approach allows us to characterize how odors are represented at the level of input to the olfactory bulb. Receptor neurons converge onto and terminate within spherical neuropile called glomeruli. Using two-photon imaging, we found that all neurons innervating a glomerulus show similar odorant response properties, confirming the idea that glomeruli act as functional units in representing odors. With wide-field imaging, we found that odorant representations tend to involve input to many, widely distributed glomeruli, and that these representations change dramatically with odorant concentration. We also found that input to glomeruli is temporally complex, with receptor neuron input showing both glomerulus- and odorant-specific kinetics. Thus, representations of odorant information in the olfactory bulb are spatially distributed, tend to involve large numbers of glomeruli, and show complex but stereotyped temporal dynamics. We hypothesize that all of these parameters are important in coding information about both odorant identity and concentration.

Genetically encoded fluorescent calcium indicator proteins (FCIPs) are promising tools to study calcium dynamics in many activity-dependent molecular and cellular processes. Great hopes—for the measurement of population activity, in particular—have therefore been placed on calcium indicators derived from the green fluorescent protein and their expression in (selected) neuronal populations. Calcium transients can rise within milliseconds, making them suitable as reporters of fast neuronal activity. We here report the production of stable transgenic mouse lines with two different functional calcium indicators, inverse pericam and camgaroo-2, under the control of the tetracycline-inducible promoter. Using a variety of in vitro and in vivo assays, we find that stimuli known to increase intracellular calcium concentration (somatically triggered action potentials (APs) and synaptic and sensory stimulation) can cause substantial and rapid changes in FCIP fluorescence of inverse pericam and camgaroo-2., Winfred Denk and colleagues succeed in generating transgenic mice that express one of two calcium indicators in their cells, creating a valuable tool to study neuronal activity

The processing of odor-evoked activity in the olfactory bulb (OB) of zebrafish was studied by extracellular single unit recordings from the input and output neurons, i.e., olfactory receptor neurons (ORNs) and mitral cells (MCs), respectively. A panel of 16 natural amino acid odors was used as stimuli. Responses of MCs, but not ORNs, changed profoundly during the first few hundred milliseconds after response onset. In MCs, but not ORNs, the total evoked excitatory activity in the population was initially odordependent but subsequently converged to a common level. Hence, the overall population activity is regulated by network interactions in the OB. The tuning widths of both ORN and MC response profiles were similar and, on average, stable over time. However, when analyzed for individual neurons, MC response profiles could sharpen (excitatory response to fewer odors) or broaden (excitatory response to more odors), whereas ORN response profiles remained nearly unchanged. Several observations indicate that dynamic inhibition plays an important role in this remodeling. Finally, the reliability of odor identification based on MC population activity patterns improved over time, whereas odor identification based on ORN activity patterns was most reliable early in the odor response. These results demonstrate that several properties of MC, but not ORN, activity change during the initial phase of the odor response with important consequences for odor-encoding activity patterns. Furthermore, our data indicate that inhibitory interactions in the OB are important in dynamically shaping the activity of OB output neurons.

The senses of hearing and balance in vertebrates rely on the sensory hair cells (HCs) of the inner ear. The central element of the HC's transduction apparatus is a mechanically gated ion channel of unknown identity. Here we report that the zebrafish ortholog of Drosophila no mechanoreceptor potential C ( nompC ), which encodes a transient receptor potential (TRP) channel, is critical for HC mechanotransduction. In zebrafish larvae, nompC is selectively expressed in sensory HCs. Morpholino-mediated removal of nompC function eliminated transduction-dependent endocytosis and electrical responses in HCs, resulting in larval deafness and imbalance. These observations indicate that nompC encodes a vertebrate HC mechanotransduction channel.

The relative importance of spatial and temporal patterning of odor-evoked activity in the olfactory bulb is controversial. Recently, voltage-sensitive dye imaging has been used to demonstrate that odor-evoked glomerular patterns evolve over time in an odor-specific manner. The results suggest that odor information is contained in a combination of spatial and temporal response components.

Bivalent peptidic thrombin inhibitors consisting of an N-terminal d-cyclohexylalanine-Pro-N(alpha)(Me)Arg active-site fragment, a flexible polyglycine linker, and a C-terminal hirugen-like segment directed towards the fibrinogen recognition exosite inhibit thrombin with K(i) values in the picomolar range, remaining stable in buffered solution at pH 7.8 for at least 15 hours. In order to investigate the structural basis of this increased stability, the most potent of these inhibitors, I-11 (K(i)=37pM), containing an N(alpha)(Me)Arg-Thr bond, was crystallized in complex with human alpha-thrombin. X-ray data were collected to 1.8A resolution and the crystal structure of this complex was determined. The Fourier map displays clear electron density for the N-terminal fragment and for the exosite binding segment. It indicates, however, that in agreement with Edman sequencing, the peptide had been cleaved in the crystal, presumably due to the long incubation time of 14 days needed for crystallization and data collection. The N(alpha)(Me) group is directed toward the carbonyl oxygen atom of Ser214, pushing the Ser195 O(gamma) atom out of its normal site. This structure suggests that upon thrombin binding, the scissile peptide bond of the intact peptide and the Ser195 O(gamma) are separated from each other, impairing the nucleophilic attack of the Ser195 O(gamma) toward the N(alpha)(Me)Arg carbonyl group. In the time-scale of two weeks, however, cleavage geometries favoured by the crystal allow catalysis at a slow rate.

The type II transmembrane multidomain serine proteinase MT-SP1/matriptase is highly expressed in many human cancer-derived cell lines and has been implicated in extracellular matrix re-modeling, tumor growth, and metastasis. We have expressed the catalytic domain of MT-SP1 and solved the crystal structures of complexes with benzamidine at 1.3 A and bovine pancreatic trypsin inhibitor at 2.9 A. MT-SP1 exhibits a trypsin-like serine proteinase fold, featuring a unique nine-residue 60-insertion loop that influences interactions with protein substrates. The structure discloses a trypsin-like S1 pocket, a small hydrophobic S2 subsite, and an open negatively charged S4 cavity that favors the binding of basic P3/P4 residues. A complementary charge pattern on the surface opposite the active site cleft suggests a distinct docking of the preceding low density lipoprotein receptor class A domain. The benzamidine crystals possess a freely accessible active site and are hence well suited for soaking small molecules, facilitating the improvement of inhibitors. The crystal structure of the MT-SP1 complex with bovine pancreatic trypsin inhibitor serves as a model for hepatocyte growth factor activator inhibitor 1, the physiological inhibitor of MT-SP1, and suggests determinants for the substrate specificity.

▪ Abstract We examine early olfactory processing in the vertebrate and insect olfactory systems, using a computational perspective. What transformations occur between the first and second olfactory processing stages? What are the causes and consequences of these transformations? To answer these questions, we focus on the functions of olfactory circuit structure and on the role of time in odor-evoked integrative processes. We argue that early olfactory relays are active and dynamical networks, whose actions change the format of odor-related information in very specific ways, so as to refine stimulus identification. Finally, we introduce a new theoretical framework (“winnerless competition”) for the interpretation of these data.

Mitral cells (MCs) in the olfactory bulb (OB) respond to odors with slow temporal firing patterns. The representation of each odor by activity patterns across the MC population thus changes continuously throughout a stimulus, in an odor-specific manner. In the zebrafish OB, we found that this distributed temporal patterning progressively reduced the similarity between ensemble representations of related odors, thereby making each odor's representation more specific over time. The tuning of individual MCs was not sharpened during this process. Hence, the individual responses of MCs did not become more specific, but the odor-coding MC assemblies changed such that their overlap decreased. This optimization of ensemble representations did not occur among olfactory afferents but resulted from OB circuit dynamics. Time can therefore gradually optimize stimulus representations in a sensory network.

How features are extracted from chemical stimuli by the sense of smell still is not clear. Presumably many more odors can be distinguished than there are receptors in the large family of odorant receptor molecules. Thus, perception of odorants seems to require a combinatorial coding strategy. We have used zebrafish, Danio rerio, as a model system to analyse the neural representation of odorants at the molecular, connectional, and physiological level. Zebrafish possess a well-developed sense of smell that governs a variety of behaviors. We find that the family 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. We observe spatial patterning of odorant receptor gene expression, of glomeruli and of odorant-induced responses in the olfactory bulb, all of which are reproducible between individuals. Response to odorants, as inferred from odorant receptor gene expression, is broadly distributed within the sensory surface. In contrast, representation of odorants is much more localized at the input level of the olfactory bulb. Classes and subclasses of odorants are represented chemotopically, whereas closely related odorants are distinguished by combinatorial encoding on the basis of broadly tuned glomerular modules.

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 F2αand 17α,20β-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.

The goal of making sense of the sense of smell has come a step closer. Work on fruitflies reveals that odorant receptors act as bidirectional chemical detectors and determine the function of sensory neurons.

Background Rag1 (Recombination activation gene-1) mediates genomic rearrangement and is essential for adaptive immunity in vertebrates. This gene is also expressed in the olfactory epithelium, but its function there is unknown. Results Using a transgenic zebrafish line and immunofluorescence, we show that Rag1 is expressed and translated in a subset of olfactory sensory neurons (OSNs). Neurons expressing GFP under the Rag1 promoter project their axons to the lateral region of the olfactory bulb only, and axons with the highest levels of GFP terminate in a single glomerular structure. A subset of GFP-expressing neurons contain Gαo, a marker for microvillous neurons. None of the GFP-positive neurons express Gαolf, Gαq or the olfactory marker protein OMP. Depletion of RAG1, by morpholino-mediated knockdown or mutation, did not affect axon targeting. Calcium imaging indicates that amino acids evoke chemotopically organized glomerular activity patterns in a Rag1 mutant. Conclusion Rag1 expression is restricted to a subpopulation of zebrafish olfactory neurons projecting to the lateral olfactory bulb. RAG1 catalytic activity is not essential for axon targeting, nor is it likely to be required for regulation of odorant receptor expression or the response of OSNs to amino acids.

Virtual realities are powerful tools to analyze and manipulate interactions between animals and their environment and to enable measurements of neuronal activity during behavior. In many species, however, optical access to the brain and/or the behavioral repertoire are limited. We developed a high-resolution virtual reality for head-restrained adult zebrafish, which exhibit cognitive behaviors not shown by larvae. We noninvasively measured activity throughout the dorsal telencephalon by multiphoton calcium imaging. Fish in the virtual reality showed regular swimming patterns and were attracted to animations of conspecifics. Manipulations of visuo-motor feedback revealed neurons that responded selectively to the mismatch between the expected and the actual visual consequences of motor output. Such error signals were prominent in multiple telencephalic areas, consistent with models of predictive processing. A virtual reality system for adult zebrafish therefore provides opportunities to analyze neuronal processing mechanisms underlying higher brain functions including decision making, associative learning, and social interactions. Complex behaviors and the underlying neural activity in adult zebrafish can be accessed through a virtual reality system in combination with two-photon microscopy.

Intelligent behavior involves associations between high-dimensional sensory representations and behaviorally relevant qualities such as valence. Learning of associations involves plasticity of excitatory connectivity, but it remains poorly understood how information flow is reorganized in networks and how inhibition contributes to this process. We trained adult zebrafish in an appetitive odor discrimination task and analyzed odor representations in a specific compartment of the posterior zone of the dorsal telencephalon (Dp), the homolog of mammalian olfactory cortex. Associative conditioning enhanced responses with a preference for the positively conditioned odor. Moreover, conditioning systematically remapped odor representations along an axis in coding space that represented attractiveness (valence). Interindividual variations in this mapping predicted variations in behavioral odor preference. Photoinhibition of interneurons resulted in specific modifications of odor representations that mirrored effects of conditioning and reduced experience-dependent, interindividual variations in odor-valence mapping. These results reveal an individualized odor-to-valence map that is shaped by inhibition and reorganized during learning.