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4. Laissue, P.P., and Vosshall, L.B. (2008). The 11. Bai, L., Goldman, A.L., and Carlson, J.R. (2009). photoreceptor subtypes in the Drosophila olfactory sensory map in Drosophila. Adv. Exp. Positive and negative regulation of odor retina. Dev. Cell 25, 93–105. Med. Biol. 628, 102–114. receptor gene choice in Drosophila by acj6. 17. Wilkins, A.S. (2002). The Evolution of 5. Goldman, A.L., Van der Goes van Naters, W., J. Neurosci. 29, 12940–12947. Developmental Pathways (Sunderland, MA: Lessing, D., Warr, C.G., and Carlson, J.R. 12. Baanannou, A., Mojica-Vazquez, L.H., Sinauer). (2005). Coexpression of two functional odor Darras, G., Couderc, J.L., Cribbs, D.L., 18. Ramdya, P., and Benton, R. (2010). Evolving receptors in one neuron. Neuron 45, 661–666. Boube, M., and Bourbon, H.M. (2013). olfactory systems on the fly. Trends Genet. 26, 6. Rodrigues, V., and Hummel, T. (2008). Drosophila distal-less and Rotund bind a 307–316. Development of the Drosophila olfactory single enhancer ensuring reliable and robust 19. Mikeladze-Dvali, T., Wernet, M.F., Pistillo, D., system. Adv. Exp. Med. Biol. 628, 82–101. bric-a-brac2 expression in distinct limb Mazzoni, E.O., Teleman, A.A., Chen, Y.W., 7. Gupta, B.P., and Rodrigues, V. (1997). Atonal is morphogenetic fields. PLoS Genet. 9, Cohen, S., and Desplan, C. (2005). The growth a proneural gene for a subset of olfactory sense e1003581. regulators warts/lats and melted interact in a organs in Drosophila. Genes Cells 2, 225–233. 13. Pearson, B.J., and Doe, C.Q. (2004). bistable loop to specify opposite fates in 8. Goulding, S.E., zur Lage, P., and Jarman, A.P. Specification of temporal identity in the Drosophila R8 photoreceptors. Cell 122, (2000). amos, a proneural gene for Drosophila developing nervous system. Annu. Rev. Cell 775–787. olfactory sense organs that is regulated by Dev. Biol. 20, 619–647. lozenge. Neuron 25, 69–78. 14. Technau, G.M., Berger, C., and Urbach, R. 9. Gupta, B.P., Flores, G.V., Banerjee, U., and (2006). Generation of cell diversity and 1Department of Neurobiology, Stanford Rodrigues, V. (1998). Patterning an epidermal segmental pattern in the embryonic central University, Stanford, CA 94305, USA. 2Center field: Drosophila lozenge, a member of the nervous system of Drosophila. Dev. Dyn. 235, for Developmental Genetics, Department of AML-1/Runt family of transcription factors, 861–869. specifies olfactory sense organ type in a 15. Song, E., de Bivort, B., Dan, C., and Kunes, S. Biology, New York University, dose-dependent manner. Dev. Biol. 203, (2012). Determinants of the Drosophila odorant 100 Washington Square East, New York, 400–411. receptor pattern. Dev Cell. 22, 363–376. NY 10003-6688, USA. 10. Tichy, A.L., Ray, A., and Carlson, J.R. (2008). 16. Thanawala, S.U., Rister, J., Goldberg, G.W., *E-mail: [email protected] A new Drosophila POU gene, pdm3, acts in Zuskov, A., Olesnicky, E.C., Flowers, J.M., odor receptor expression and axon targeting Jukam, D., Purugganan, M.D., Gavis, E.R., of olfactory neurons. J. Neurosci. 28, Desplan, C., et al. (2013). Regional modulation 7121–7129. of a stochastically expressed factor determines http://dx.doi.org/10.1016/j.cub.2013.10.050

The latter strategy can be achieved by Olfactory Neuroscience: dividing the activity of each neuron by the summed activity over a pool of Normalization Is the Norm neurons, a computation known as normalization [5]. Normalization has been described in A recent study shows that neural circuits from vertebrates and invertebrates several sensory processing circuits use common strategies to stabilize odor representations across a wide range near the sensory periphery, where it of concentrations. controls for stimulus intensity [5]. It also occurs in many cortical regions, Elizabeth J. Hong of perceptible natural sounds. including higher processing regions, and Rachel I. Wilson Similarly, perceived odor quality where it controls for more complex can remain constant over many properties of sensory stimuli. For Neural systems must constantly orders of magnitude [2]. example, in visual object recognition re-adjust their sensitivity as their Large variations in stimulus intensity areas, normalization controls for the inputs fluctuate. How this process is pose a problem. The total activity in number of visual objects in a scene, so implemented in the brain is poorly any sensory brain region should not be that neural activity is more closely understood. In a new study, Zhu, Frank, allowed to vary over a large range. related to the nature of the objects than and Friedrich [1] describe a mechanism One reason is that neural activity is to their total number [6]. Indeed, in the zebrafish for extremely costly, from a metabolic normalization has been proposed to be equalizing odor-evoked activity across perspective [3]. A second reason is that a basic building block of neural a wide range of odor concentrations. neurons have a finite capacity for computations. For this reason, there is Notably, both lateral inhibition and information transmission, and so the interest in its underlying mechanisms. lateral excitation play a role in adjusting more information they transmit about Synaptic inhibition is a logical neural sensitivity. This study reveals intensity, the less capacity is available candidate, but the evidence for this idea remarkable parallels between the to transmit anything else [2]. has often been indirect or mixed [5]. zebrafish olfactory bulb and the Ultimately, the perceptual quality of a Drosophila antennal lobe, where lateral stimulus is largely robust to changing Normalization in Fish Olfaction inhibition and lateral excitation also stimulus intensity — for example, a The new study by Zhu et al. [1] takes a work together to equalize neural tasty food smells equally delicious, big stride toward understanding the activity. whether it is under our nose or across mechanisms of normalization. the street. Using calcium imaging to monitor Normalization: A Canonical Neural Neural systems can respond to this odor-evoked activity in principal Computation problem by turning down their gain neurons of the olfactory bulb (mitral Natural sensory stimuli vary when stimulus intensity grows. This cells), they found that total levels of enormously in intensity. For example, sort of process is often known as activity were remarkably invariant to the luminance of perceptible adaptation or gain control [2,4]. Each odor concentration over a 10,000-fold natural visual images can vary over neuron can control its own gain, or it range. This result suggests that mitral 10-billion fold, as can the intensity can take advice from other neurons. cell activity is being normalized. Current Biology Vol 23 No 24 R1092

ABC quite different from the conventional Danio rerio Drosophila melanogaster view of normalization, where the effects Glomerulus Control of a normalization pool are assumed to Olfactory Olfactory bulb Olfactory Antennal lobe receptor mitral receptor projection be purely inhibitory. neuron cell neuron neuron Silence dlx4/6 and

Firing rate (Hz) block gap junctions Fish and Fly Excitatory local Log concentration Another notable feature of this story is interneuron dlx4/6 its connection to studies of the neurons Inhibitory Drosophila antennal lobe. It has long local Control interneuron been noted that the olfactory bulb and antennal lobe share a common Firing rate (Hz) Block gap junctions feedforward architecture (Figure 1), Log concentration and we are now learning that these Current Biology circuits share much more. In the Drosophila antennal lobe, as in the Figure 1. Fish and flies share common circuit mechanisms to stabilize olfactory responses. olfactory bulb, glomeruli are (A) Schematic of the zebrafish olfactory bulb. All the olfactory receptor neurons that express interconnected via local interneurons. the same odorant receptor gene project to the same glomerulus [18], and most individual Most of these are GABAergic and their mitral cells receive direct olfactory receptor neuron input from a single glomerulus [19]. processes extend across many Glomeruli are interconnected by local interneurons, including a population of GABAergic glomeruli. It was recently shown that cells called dlx4/6 neurons. Zhu et al. [1] show that the dlx4/6 neurons are electrically coupled to mitral cells, in addition to forming GABAergic synapses. As a consequence, these GABAergic interneurons they have both excitatory and inhibitory effects on mitral cells. Whether dlx4/6 neurons normalize activity in antennal lobe inhibit mitral cells directly or indirectly is uncertain. (B) Schematic of the fruit fly antennal projection neurons (the analog lobe. The feedforward architecture of this circuit is essentially the same as in the olfactory of mitral cells). Normalization tends to bulb [20]. Moreover, recent studies have shown that glomeruli are interconnected by both equalize the total amount of activity in inhibitory local neurons and excitatory local neurons. Excitatory local neurons make electrical Drosophila projection neurons across a connections with antennal lobe projection neurons [11–13]. Thus, in both fish and flies, inhibitory interactions between glomeruli are mediated by chemical synapses, whereas excit- range of odor concentrations [8–10]. atory interactions between glomeruli are mediated by electrical synapses. (C) Cartoon of This is similar to what Zhu et al. [1] have concentration–response functions in mitral cells. The total firing rate of all mitral cells varies now shown in fish. little with odor concentration in control conditions. When dlx4/6 neurons are silenced opto- Moreover, recent studies also genetically, and gap junctions are also blocked pharmacologically, the slope of this function revealed that the Drosophila antennal becomes steeper (top). Simply blocking gap junctions diminishes responses to low odor lobe contains some interneurons that concentrations, but has no effect on responses to high concentrations (bottom). (Adapted with permission from [1].) make electrical connections with projection neurons. Like the inhibitory interneurons, these cells also extend When Zhu et al. [1] measured activity neurons elicited not only inhibition in their processes across many in a genetically defined population of mitral cells, but also excitation. glomeruli. This circuit produces lateral GABAergic interneurons (termed Pharmacological experiments excitation which can boost the odor dlx4/6 neurons), they found something indicated that the excitation is due to a responses of Drosophila projection quite different. Namely, total activity in direct electrical connection between neurons, especially when these odor dlx4/6 neurons rose linearly with the dlx4/6 neurons and mitral cells. In sum, responses are weak [11–13]. This is logarithm of odor concentration. This activation of dlx4/6 neurons produces remarkably similar to the new results suggests that dlx4/6 neurons receive competing effects: it puts on the brakes in fish. direct input from the nose via olfactory (via GABAergic chemical synapses) It is curious that in both fish and fly, receptor neurons, the summed activity and also hits the gas (via electrical lateral excitation is implemented via of which also grows roughly linearly synapses). Which effect wins? electrical synapses, not chemical with log concentration [7]. To address this question, Zhu et al. synapses. Why? It is tempting to The next step was to test the effect [1] used a two-pronged manipulation: speculate that gap junctions are useful on mitral cells of activating the dlx4/6 they blocked gap junctions in this context because electrical neurons. Optogenetic activation of pharmacologically (to block electrical synapses (unlike chemical synapses) dlx4/6 neurons elicited inhibitory output), and they also hyperpolarized are fundamentally conservative: the synaptic conductances in mitral cells, the dlx4/6 neurons optogenetically current that flows into one cell is as expected, given that the dlx4/6 (to block chemical output). This necessarily flowing out of another cell, neurons are GABAergic. Taken manipulation increased mitral cell meaning total current is conserved. In together, these results suggest that responses to high-concentration essence, an electrical connection dlx4/6 neurons collectively encode odors, and it decreased responses to between two neurons causes those odor concentration, and that they low-concentration odors. neurons to (slightly) average out their inhibit mitral cells as concentration Taken together, these results show respective activity levels, keeping total grows, thereby normalizing mitral that excitation dominates at low odor activity constant. This feature of cell activity. concentrations, but inhibition electrical synapses might help prevent dominates at high concentrations. Both lateral excitation from spreading Hitting the Brakes — and the Gas effects are important for the role of uncontrollably throughout the circuit. Here the tale takes a strange turn. dlx4/6 neurons in equalizing activity to Could a similar mechanism operate Optogenetic activation of dlx4/6 a wide range of concentrations. 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Ultrastructural and dye filling studies of molecular biology in the 20th century 10. Olsen, S.R., and Wilson, R.I. (2008). Lateral presynaptic inhibition mediates gain control in have suggested that GABAergic hinged on our ability to spot an olfactory circuit. Nature 452, 956–960. interneurons make gap junctions onto homologies between amino acid 11. Yaksi, E., and Wilson, R.I. (2010). Electrical mitral cells in the mammalian bulb sequences in different protein coupling between olfactory glomeruli. Neuron 67, 1034–1047. [14,15]. However, this remains a domains, and to grasp the systematic 12. Huang, J., Zhang, W., Qiao, W., Hu, A., and controversial topic that requires relationship between the primary Wang, Z. (2010). Functional connectivity and selective odor responses of excitatory local further work. structure of a domain and its function. interneurons in Drosophila antennal lobe. Many such molecular modules are now Neuron 67, 1021–1033. New Questions known to reoccur throughout the 13. Olsen, S.R., Bhandawat, V., and Wilson, R.I. (2007). Excitatory interactions between The study by Zhu et al. [1] raises many animal kingdom. Being able to spot olfactory processing channels in the Drosophila questions. First, exactly which similar kinds of structure–function antennal lobe. Neuron 54, 89–103. 14. Kosaka, T., and Kosaka, K. (2003). Neuronal interneurons are involved? The dlx4/6 relationships in neural circuits would gap junctions in the rat main olfactory bulb, neurons are a heterogeneous accelerate discovery in neuroscience. with special reference to intraglomerular gap population [1], like the interneurons of For this reason, comparative biology junctions. Neurosci. Res. 45, 189–209. 15. Paternostro, M.A., Reyher, C.K., and the Drosophila antennal lobe, and remains essential to the search for Brunjes, P.C. (1995). Intracellular injections of some of these neurons may play general principles. lucifer yellow into lightly fixed mitral cells reveal neuronal dye-coupling in the entirely distinct roles. Identifying a developing rat olfactory bulb. Dev. Brain Res. genetic marker for each relevant 84, 1–10. References interneuron type would permit more 16. Shipley, M.T., and Ennis, M. (1996). Functional 1. Zhu, P., Frank, T., and Friedrich, R.W. (2013). organization of olfactory system. J. Neurobiol. targeted recordings, better mapping of Equalization of odor representations by a 30, 123–176. connectivity, and more precise network of electrically coupled inhibitory 17. Root, C.M., Masuyama, K., Green, D.S., interneurons. Nat Neurosci. 16, 1678–1686. Enell, L.E., Nassel, D.R., Lee, C.H., and manipulations. It is also notable that 2. Fairhall, A. (2014). Adaptation and natural Wang, J.W. (2008). A presynaptic gain control there are likely many other GABAergic stimulus statistics. In The Cognitive mechanism fine-tunes olfactory behavior. Neurosciences 5th Edition, M.S. Gazzaniga, ed. interneurons in the fish olfactory bulb in Neuron 59, 311–321. (Cambridge, MA: MIT Press). 18. Yoshihara, Y. (2009). Molecular genetic addition to the dlx4/6 neurons. In the 3. Laughlin, S.B. (2001). Energy as a constraint on dissection of the zebrafish olfactory system. In mammalian bulb there are several the coding and processing of sensory Chemosensory Systems in Mammals, Fishes, information. Curr. Opin. Neurobiol. 11, 475–480. and , W. Meyerhof and S.I. Korsching, populations of periglomerular cells in 4. Shapley, R., and Enroth-Cugell, C. (1984). eds. (Heidelberg Berlin: Springer-Verlag), the superficial glomerular layer, as well Visual adaptation and retinal gain controls. pp. 97–120. Prog. Retin. Res. 3, 263–346. as deep-layer granule cells, which 19. Fuller, C.L., Yettaw, H.K., and Byrd, C.A. (2006). 5. Carandini, M., and Heeger, D.J. (2012). Mitral cells in the olfactory bulb of adult outnumber all other cell types in the Normalization as a canonical neural zebrafish (Danio rerio): morphology and bulb by at least an order of magnitude computation. Nat. Rev. Neurosci. 13, 51–62. distribution. J. Comp. Neurol. 499, 218–230. 6. Zoccolan, D., Cox, D.D., and DiCarlo, J.J. 20. Wilson, R.I. (2013). Early olfactory processing in [16]. What specific roles do each of (2005). Multiple object response normalization Drosophila: mechanisms and principles. Annu. these different inhibitory circuits play, in monkey inferotemporal cortex. J. Neurosci. Rev. Neurosci. 36, 217–241. 25, 8150–8164. and how do they interact? 7. Friedrich, R.W., and Korsching, S.I. (1997). Second, do specific odor stimuli Combinatorial and chemotopic odorant coding elicit specific spatial patterns of in the zebrafish olfactory bulb visualized by Department of Neurobiology, Harvard optical imaging. Neuron 18, 737–752. Medical School, 220 Longwood Ave., Boston, interactions among olfactory 8. Olsen, S.R., Bhandawat, V., and Wilson, R.I. MA 02115, USA. glomeruli? Or alternatively, is this a (2010). Divisive normalization in olfactory population codes. Neuron 66, 287–299. E-mail: [email protected], global interaction that simply scales in 9. Asahina, K., Louis, M., Piccinotti, S., and [email protected] strength with the total level of afferent Vosshall, L.B. (2009). A circuit supporting concentration-invariant odor perception in input to the circuit? None of the studies Drosophila. J. Biol. 8,9. http://dx.doi.org/10.1016/j.cub.2013.10.056 discussed here provides a clear answer to this question. This issue is critical to understanding how interneurons affect olfactory processing. Third, why does this circuit have such diverse effects on different target Evolution: Unveiling Early Alveolates neurons? When the dlx4/6 neurons were manipulated, there were large The isolation and characterisation of a novel protist lineage enables the variations across cells in the effects reconstruction of early evolutionary events that gave rise to ciliates, malaria this had on neural activity. For a given parasites, and coral symbionts. These events include dramatic changes in odor stimulus, some cells were mitochondrial genome content and organisation. inhibited, but others were disinhibited [1]. Again, this finding has a parallel in the Drosophila antennal lobe, where Richard G. Dorrell1, extremely important functions in identified olfactory glomeruli have Erin R. Butterfield1,2, planetary ecology, or are major diverse levels of sensitivity to lateral R. Ellen R. Nisbet1,2, human pathogens [1]. Some protists inhibition and lateral excitation and Christopher J. Howe1,* have served as models for processes [8,13,17]. What are the mechanisms of broad biological significance; and significance of this diversity? Animals, plants, and other multicellular for example, early work on telomere Finally, how deep are the functional organisms are a drop in the ocean maintenance used the ciliate and structural similarities between of eukaryotic diversity. A vast array Tetrahymena [2]. However, compared neural circuits in different organisms? of different protist lineages are to animals and other model We should remember that the success known, many of which have eukaryotes, most of the cell biology of