Developmental and evolutionary constraints on olfactory circuit selection.

SignificanceIn this work, we explore the hypothesis that biological neural networks optimize their architecture, through evolution, for learning. We study early olfactory circuits of mammals and insects, which have relatively similar structure but a huge diversity in size. We approximate these circuits as three-layer networks and estimate, analytically, the scaling of the optimal hidden-layer size with input-layer size. We find that both longevity and information in the genome constrain the hidden-layer size, so a range of allometric scalings is possible. However, the experimentally observed allometric scalings in mammals and insects are consistent with biologically plausible values. This analysis should pave the way for a deeper understanding of both biological and artificial networks.

Cellular bases of olfactory circuit assembly revealed by systematic time-lapse imaging.

Neural circuit assembly features simultaneous targeting of numerous neuronal processes from constituent neuron types, yet the dynamics is poorly understood. Here, we use the Drosophila olfactory circuit to investigate dynamic cellular processes by which olfactory receptor neurons (ORNs) target axons precisely to specific glomeruli in the ipsi- and contralateral antennal lobes. Time-lapse imaging of individual axons from 30 ORN types revealed a rich diversity in extension speed, innervation timing, and ipsilateral branch locations and identified that ipsilateral targeting occurs via stabilization of transient interstitial branches. Fast imaging using adaptive optics-corrected lattice light-sheet microscopy showed that upon approaching target, many ORN types exhibiting "exploring branches" consisted of parallel microtubule-based terminal branches emanating from an F-actin-rich hub. Antennal nerve ablations uncovered essential roles for bilateral axons in contralateral target selection and for ORN axons to facilitate dendritic refinement of postsynaptic partner neurons. Altogether, these observations provide cellular bases for wiring specificity establishment.

Chronic chemogenetic stimulation of the anterior olfactory nucleus reduces newborn neuron survival in the adult mouse olfactory bulb.

During adult rodent life, newborn neurons are added to the olfactory bulb (OB) in a tightly controlled manner. Upon arrival in the OB, input synapses from the local bulbar network and the higher olfactory cortex precede the formation of functional output synapses, indicating a possible role for these regions in newborn neuron survival. An interplay between the environment and the piriform cortex in the regulation of newborn neuron survival has been suggested. However, the specific network and the neuronal cell types responsible for this effect have not been elucidated. Furthermore, the role of the other olfactory cortical areas in this process is not known. Here we demonstrate that pyramidal neurons in the mouse anterior olfactory nucleus, the first cortical area for odor processing, have a key role in the survival of newborn neurons. Using DREADD (Designer Receptors Exclusively Activated by Designer Drugs) technology, we applied chronic stimulation to the anterior olfactory nucleus and observed a decrease in newborn neurons in the OB through induction of apoptosis. These findings provide further insight into the network regulating neuronal survival in adult neurogenesis and strengthen the importance of the surrounding network for sustained integration of new neurons.

Generation and Characterization of a Cell Type-Specific, Inducible Cre-Driver Line to Study Olfactory Processing.

In sensory systems of the brain, mechanisms exist to extract distinct features from stimuli to generate a variety of behavioral repertoires. These often correspond to different cell types at various stages in sensory processing. In the mammalian olfactory system, complex information processing starts in the olfactory bulb, whose output is conveyed by mitral cells (MCs) and tufted cells (TCs). Despite many differences between them, and despite the crucial position they occupy in the information hierarchy, Cre-driver lines that distinguish them do not yet exist. Here, we sought to identify genes that are differentially expressed between MCs and TCs of the mouse, with an ultimate goal to generate a cell type-specific Cre-driver line, starting from a transcriptome analysis using a large and publicly available single-cell RNA-seq dataset (Zeisel et al., 2018). Many genes were differentially expressed, but only a few showed consistent expressions in MCs and at the specificity required. After further validating these putative markers using ISH, two genes (i.e., Pkib and Lbdh2) remained as promising candidates. Using CRISPR/Cas9-mediated gene editing, we generated Cre-driver lines and analyzed the resulting recombination patterns. This indicated that our new inducible Cre-driver line, Lbhd2-CreERT2, can be used to genetically label MCs in a tamoxifen dose-dependent manner, both in male and female mice, as assessed by soma locations, projection patterns, and sensory-evoked responses in vivo Hence, this is a promising tool for investigating cell type-specific contributions to olfactory processing and demonstrates the power of publicly accessible data in accelerating science.SIGNIFICANCE STATEMENT In the brain, distinct cell types play unique roles. It is therefore important to have tools for studying unique cell types specifically. For the sense of smell in mammals, information is processed first by circuits of the olfactory bulb, where two types of cells, mitral cells and tufted cells, output different information. We generated a transgenic mouse line that enables mitral cells to be specifically labeled or manipulated. This was achieved by looking for genes that are specific to mitral cells using a large and public gene expression dataset, and creating a transgenic mouse using the gene editing technique, CRISPR/Cas9. This will allow scientists to better investigate parallel information processing underlying the sense of smell.

Olfaction in Lamprey Pallium Revisited-Dual Projections of Mitral and Tufted Cells.

The presence of two separate afferent channels from the olfactory glomeruli to different targets in the brain is unravelled in the lamprey. The mitral-like cells send axonal projections directly to the piriform cortex in the ventral part of pallium, whereas the smaller tufted-like cells project separately and exclusively to a relay nucleus called the dorsomedial telencephalic nucleus (dmtn). This nucleus, located at the interface between the olfactory bulb and pallium, in turn projects to a circumscribed area in the anteromedial, ventral part of pallium. The tufted-like cells are activated with short latency from the olfactory nerve and terminate with mossy fibers on the dmtn cells, wherein they elicit large unitary excitatory postsynaptic potentials (EPSPs). In all synapses along this tufted-like cell pathway, there is no concurrent inhibition, in contrast to the mitral-like cell pathway. This is similar to recent findings in rodents establishing two separate exclusive projection patterns, suggesting an evolutionarily conserved organization.

The central nervous system of whip spiders (Amblypygi): Large mushroom bodies receive olfactory and visual input.

Whip spiders (Amblypygi) are known for their nocturnal navigational abilities, which rely on chemosensory and tactile cues and, to a lesser degree, on vision. Unlike true spiders, the first pair of legs in whip spiders is modified into extraordinarily long sensory organs (antenniform legs) covered with thousands of mechanosensory, olfactory, and gustatory sensilla. Olfactory neurons send their axons through the leg nerve into the corresponding neuromere of the central nervous system, where they terminate on a particularly large number (about 460) of primary olfactory glomeruli, suggesting an advanced sense of smell. From the primary glomeruli, olfactory projection neurons ascend to the brain and terminate in the mushroom body calyx on a set of secondary olfactory glomeruli, a feature that is not known from olfactory pathways of other animals. Another part of the calyx receives visual input from the secondary visual neuropil (the medulla). This calyx region is composed of much smaller glomeruli ("microglomeruli"). The bimodal input and the exceptional size of their mushroom bodies may support the navigational capabilities of whip spiders. In addition to input to the mushroom body, we describe other general anatomical features of the whip spiders' central nervous system.

Antennal-lobe neurons in the moth Helicoverpa armigera: Morphological features of projection neurons, local interneurons, and centrifugal neurons.

The relatively large primary olfactory center of the insect brain, the antennal lobe (AL), contains several heterogeneous neuronal types. These include projection neurons (PNs), providing olfactory information to higher-order neuropils via parallel pathways, and local interneurons (LNs), which provide lateral processing within the AL. In addition, various types of centrifugal neurons (CNs) offer top-down modulation onto the other AL neurons. By performing iontophoretic intracellular staining, we collected a large number of AL neurons in the moth, Helicoverpa armigera, to examine the distinct morphological features of PNs, LNs, and CNs. We characterize 190 AL neurons. These were allocated to 25 distinct neuronal types or sub-types, which were reconstructed and placed into a reference brain. In addition to six PN types comprising 15 sub-types, three LN and seven CN types were identified. High-resolution confocal images allowed us to analyze AL innervations of the various reported neurons, which demonstrated that all PNs innervating ventroposterior glomeruli contact a protocerebral neuropil rarely targeted by other PNs, that is the posteriorlateral protocerebrum. We also discuss the functional roles of the distinct CNs, which included several previously uncharacterized types, likely involved in computations spanning from multisensory processing to olfactory feedback signalization into the AL.

Convergence of Olfactory Inputs within the Central Nervous System of a Cartilaginous and a Bony Fish: An Anatomical Indicator of Olfactory Sensitivity.

The volume of the olfactory bulbs (OBs) relative to the brain has been used previously as a proxy for olfactory capabilities in many vertebrate taxa, including fishes. Although this gross approach has predictive power, a more accurate assessment of the number of afferent olfactory inputs and the convergence of this information at the level of the telencephalon is critical to our understanding of the role of olfaction in the behaviour of fishes. In this study, we used transmission electron microscopy to assess the number of first-order axons within the olfactory nerve (ON) and the number of second-order axons in the olfactory peduncle (OP) in established model species within cartilaginous (brownbanded bamboo shark, Chiloscyllium punctatum [CP]) and bony (common goldfish, Carassius auratus [CA]) fishes. The total number of axons varied from a mean of 18.12 ± 7.50 million in the ON to a mean of 0.38 ± 0.21 million in the OP of CP, versus 0.48 ± 0.16 million in the ON and 0.09 ± 0.02 million in the OP of CA. This resulted in a convergence ratio of approximately 50:1 and 5:1, respectively, for these two species. Based on astroglial ensheathing, axon type (unmyelinated [UM] and myelinated [M]) and axon size, we found no differentiated tracts in the OP of CP, whereas a lateral and a medial tract (both of which could be subdivided into two bundles or areas) were identified for CA, as previously described. Linear regression analyses revealed significant differences not only in axon density between species and locations (nerves and peduncles), but also in axon type and axon diameter (p < 0.05). However, UM axon diameter was larger in the OPs than in the nerve in both species (p = 0.005), with no significant differences in UM axon diameter in the ON (p = 0.06) between species. This study provides an in-depth analysis of the neuroanatomical organisation of the ascending olfactory pathway in two fish taxa and a quantitative anatomical comparison of the summation of olfactory information. Our results support the assertion that relative OB volume is a good indicator of the level of olfactory input and thereby a proxy for olfactory capabilities.

Subpopulations of Projection Neurons in the Olfactory Bulb.

Generation of neuronal diversity is a biological strategy widely used in the brain to process complex information. The olfactory bulb is the first relay station of olfactory information in the vertebrate central nervous system. In the olfactory bulb, axons of the olfactory sensory neurons form synapses with dendrites of projection neurons that transmit the olfactory information to the olfactory cortex. Historically, the olfactory bulb projection neurons have been classified into two populations, mitral cells and tufted cells. The somata of these cells are distinctly segregated within the layers of the olfactory bulb; the mitral cells are located in the mitral cell layer while the tufted cells are found in the external plexiform layer. Although mitral and tufted cells share many morphological, biophysical, and molecular characteristics, they differ in soma size, projection patterns of their dendrites and axons, and odor responses. In addition, tufted cells are further subclassified based on the relative depth of their somata location in the external plexiform layer. Evidence suggests that different types of tufted cells have distinct cellular properties and play different roles in olfactory information processing. Therefore, mitral and different types of tufted cells are considered as starting points for parallel pathways of olfactory information processing in the brain. Moreover, recent studies suggest that mitral cells also consist of heterogeneous subpopulations with different cellular properties despite the fact that the mitral cell layer is a single-cell layer. In this review, we first compare the morphology of projection neurons in the olfactory bulb of different vertebrate species. Next, we explore the similarities and differences among subpopulations of projection neurons in the rodent olfactory bulb. We also discuss the timing of neurogenesis as a factor for the generation of projection neuron heterogeneity in the olfactory bulb. Knowledge about the subpopulations of olfactory bulb projection neurons will contribute to a better understanding of the complex olfactory information processing in higher brain regions.

CCKergic Tufted Cells Differentially Drive Two Anatomically Segregated Inhibitory Circuits in the Mouse Olfactory Bulb.

Delineation of functional synaptic connections is fundamental to understanding sensory processing. Olfactory signals are synaptically processed initially in the olfactory bulb (OB) where neural circuits are formed among inhibitory interneurons and the output neurons mitral cells (MCs) and tufted cells (TCs). TCs function in parallel with but differently from MCs and are further classified into multiple subpopulations based on their anatomic and functional heterogeneities. Here, we combined optogenetics with electrophysiology to characterize the synaptic transmission from a subpopulation of TCs, which exclusively express the neuropeptide cholecystokinin (CCK), to two groups of spatially segregated GABAergic interneurons, granule cells (GCs) and glomerular interneurons in mice of both sexes with four major findings. First, CCKergic TCs receive direct input from the olfactory sensory neurons (OSNs). This monosynaptic transmission exhibits high fidelity in response to repetitive OSN input. Second, CCKergic TCs drive GCs through two functionally distinct types of monosynaptic connections: (1) dendrodendritic synapses onto GC distal dendrites via their lateral dendrites in the superficial external plexiform layer (EPL); (2) axodendritic synapses onto GC proximal dendrites via their axon collaterals or terminals in the internal plexiform layer (IPL) on both sides of each bulb. Third, CCKergic TCs monosynaptically excite two subpopulations of inhibitory glomerular interneurons via dendrodendritic synapses. Finally, sniff-like patterned activation of CCKergic TCs induces robust frequency-dependent depression of the dendrodendritic synapses but facilitation of the axodendritic synapses. These results demonstrated important roles of the CCKergic TCs in olfactory processing by orchestrating OB inhibitory activities.SIGNIFICANCE STATEMENT Neuronal morphology and organization in the olfactory bulb (OB) have been extensively studied, however, the functional operation of neuronal interactions is not fully understood. We combined optogenetic and electrophysiological approaches to investigate the functional operation of synaptic connections between a specific population of excitatory output neuron and inhibitory interneurons in the OB. We found that these output neurons formed distinct types of synapses with two populations of spatially segregated interneurons. The functional characteristics of these synapses vary significantly depending on the presynaptic compartments so that these output neurons can dynamically rebalance inhibitory feedback or feedforward to other neurons types in the OB in response to dynamic rhythmic inputs.

GABAergic Input From the Basal Forebrain Promotes the Survival of Adult-Born Neurons in the Mouse Olfactory Bulb.

A unique feature of the olfactory system is the continuous generation and integration of new neurons throughout adulthood. Adult-born neuron survival and integration is dependent on activity and sensory experience, which is largely mediated by early synaptic inputs that adult-born neurons receive upon entering the olfactory bulb (OB). As in early postnatal development, the first synaptic inputs onto adult-born neurons are GABAergic. However, the specific sources of early synaptic GABA and the influence of specific inputs on adult-born neuron development are poorly understood. Here, we use retrograde and anterograde viral tracing to reveal robust GABAergic projections from the basal forebrain horizontal limb of the diagonal band of Broca (HDB) to the granule cell layer (GCL) and glomerular layer (GL) of the mouse OB. Whole-cell electrophysiological recordings indicate that these projections target interneurons in the GCL and GL, including adult-born granule cells (abGCs). Recordings from birth-dated abGCs reveal a developmental time course in which HDB GABAergic input onto abGCs emerges as the neurons first enter the OB, and strengthens throughout the critical period of abGC development. Finally, we show that removing GABAergic signaling from HDB neurons results in decreased abGC survival. Together these data show that GABAergic projections from the HDB synapse onto immature abGCs in the OB to promote their survival through the critical period, thus representing a source of long-range input modulating plasticity in the adult OB.

Bilateral and unilateral odor processing and odor perception.

Imagine smelling a novel perfume with only one nostril and then smelling it again with the other nostril. Clearly, you can tell that it is the same perfume both times. This simple experiment demonstrates that odor information is shared across both hemispheres to enable perceptual unity. In many sensory systems, perceptual unity is believed to be mediated by inter-hemispheric connections between iso-functional cortical regions. However, in the olfactory system, the underlying neural mechanisms that enable this coordination are unclear because the two olfactory cortices are not topographically organized and do not seem to have homotypic inter-hemispheric mapping. This review presents recent advances in determining which aspects of odor information are processed unilaterally or bilaterally, and how odor information is shared across the two hemispheres. We argue that understanding the mechanisms of inter-hemispheric coordination can provide valuable insights that are hard to achieve when focusing on one hemisphere alone.

Interhemispheric asymmetry of c-Fos expression in glomeruli and the olfactory tubercle following repeated odor stimulation.

Odor adaptation allows the olfactory system to regulate sensitivity to different stimulus intensities, which is essential for preventing saturation of the cell-transducing machinery and maintaining high sensitivity to persistent and repetitive odor stimuli. Although many studies have investigated the structure and mechanisms of the mammalian olfactory system that responds to chemical sensation, few studies have considered differences in neuronal activation that depend on the manner in which the olfactory system is exposed to odorants, or examined activity patterns of olfactory-related regions in the brain under different odor exposure conditions. To address these questions, we designed three different odor exposure conditions that mimicked diverse odor environments and analyzed c-Fos-expressing cells (c-Fos+ cells) in the odor columns of the olfactory bulb (OB). We then measured differences in the proportions of c-Fos-expressing cell types depending on the odor exposure condition. Surprisingly, under the specific odor condition in which the olfactory system was repeatedly exposed to the odorant for 1 min at 5-min intervals, one of the lateral odor columns and the ipsilateral hemisphere of the olfactory tubercle had more c-Fos+ cells than the other three odor columns and the contralateral hemisphere of the olfactory tubercle. However, this interhemispheric asymmetry of c-Fos expression was not observed in the anterior piriform cortex. To confirm whether the anterior olfactory nucleus pars externa (AONpE), which connects the left and right OB, contributes to this asymmetry, AONpE-lesioned mice were analyzed under the specific odor exposure condition. Asymmetric c-Fos expression was not observed in the OB or the olfactory tubercle. These data indicate that the c-Fos expression patterns of the olfactory-related regions in the brain are influenced by the odor exposure condition and that asymmetric c-Fos expression in these regions was observed under a specific odor exposure condition due to synaptic linkage via the AONpE.

Olfactory receptor and circuit evolution promote host specialization.

The evolution of animal behaviour is poorly understood1,2. Despite numerous correlations between interspecific divergence in behaviour and nervous system structure and function, demonstrations of the genetic basis of these behavioural differences remain rare3-5. Here we develop a neurogenetic model, Drosophila sechellia, a species that displays marked differences in behaviour compared to its close cousin Drosophila melanogaster6,7, which are linked to its extreme specialization on noni fruit (Morinda citrifolia)8-16. Using calcium imaging, we identify olfactory pathways in D. sechellia that detect volatiles emitted by the noni host. Our mutational analysis indicates roles for different olfactory receptors in long- and short-range attraction to noni, and our cross-species allele-transfer experiments demonstrate that the tuning of one of these receptors is important for species-specific host-seeking. We identify the molecular determinants of this functional change, and characterize their evolutionary origin and behavioural importance. We perform circuit tracing in the D. sechellia brain, and find that receptor adaptations are accompanied by increased sensory pooling onto interneurons as well as species-specific central projection patterns. This work reveals an accumulation of molecular, physiological and anatomical traits that are linked to behavioural divergence between species, and defines a model for investigating speciation and the evolution of the nervous system.

Multiple network properties overcome random connectivity to enable stereotypic sensory responses.

Connections between neuronal populations may be genetically hardwired or random. In the insect olfactory system, projection neurons of the antennal lobe connect randomly to Kenyon cells of the mushroom body. Consequently, while the odor responses of the projection neurons are stereotyped across individuals, the responses of the Kenyon cells are variable. Surprisingly, downstream of Kenyon cells, mushroom body output neurons show stereotypy in their responses. We found that the stereotypy is enabled by the convergence of inputs from many Kenyon cells onto an output neuron, and does not require learning. The stereotypy emerges in the total response of the Kenyon cell population using multiple odor-specific features of the projection neuron responses, benefits from the nonlinearity in the transfer function, depends on the convergence:randomness ratio, and is constrained by sparseness. Together, our results reveal the fundamental mechanisms and constraints with which convergence enables stereotypy in sensory responses despite random connectivity.

Cell-Surface Proteomic Profiling in the Fly Brain Uncovers Wiring Regulators.

Molecular interactions at the cellular interface mediate organized assembly of single cells into tissues and, thus, govern the development and physiology of multicellular organisms. Here, we developed a cell-type-specific, spatiotemporally resolved approach to profile cell-surface proteomes in intact tissues. Quantitative profiling of cell-surface proteomes of Drosophila olfactory projection neurons (PNs) in pupae and adults revealed global downregulation of wiring molecules and upregulation of synaptic molecules in the transition from developing to mature PNs. A proteome-instructed in vivo screen identified 20 cell-surface molecules regulating neural circuit assembly, many of which belong to evolutionarily conserved protein families not previously linked to neural development. Genetic analysis further revealed that the lipoprotein receptor LRP1 cell-autonomously controls PN dendrite targeting, contributing to the formation of a precise olfactory map. These findings highlight the power of temporally resolved in situ cell-surface proteomic profiling in discovering regulators of brain wiring.

Co-localization of two-color rAAV2-retro confirms the dispersion characteristics of efferent projections of mitral cells in mouse accessory olfactory bulb.

The accessory olfactory bulb (AOB), located at the posterior dorsal aspect of the main olfactory bulb (MOB), is the first brain relay of the accessory olfactory system (AOS), which can parallelly detect and process volatile and nonvolatile social chemosignals and mediate different sexual and social behaviors with the main olfactory system (MOS). However, due to its anatomical location and absence of specific markers, there is a lack of research on the internal and external neural circuits of the AOB. This issue was addressed by single-color labeling and fluorescent double labeling using retrograde rAAVs injected into the bed nucleus of the stria terminalis (BST), anterior cortical amygdalar area (ACo), medial amygdaloid nucleus (MeA), and posteromedial cortical amygdaloid area (PMCo) in mice. We demonstrated the effectiveness of this AOB projection neuron labeling method and showed that the mitral cells of the AOB exhibited efferent projection dispersion characteristics similar to those of the MOB. Moreover, there were significant differences in the number of neurons projected to different brain regions, which indicated that each mitral cell in the AOB could project to a different number of neurons in different cortices. These results provide a circuitry basis to help understand the mechanism by which pheromone information is encoded and decoded in the AOS.

Centrifugal projections to the main olfactory bulb revealed by transsynaptic retrograde tracing in mice.

A wide range of evidence indicates that olfactory perception is strongly involved in food intake. However, the polysynaptic circuitry linking the brain areas involved in feeding behavior to the olfactory regions is not well known. The aim of this article was to examine such circuits. Thus, we described, using hodological tools such as transsynaptic viruses (PRV152) transported in a retrograde manner, the long-distance indirect projections (two to three synapses) onto the main olfactory bulb (MOB). The ß-subunit of the cholera toxin which is a monosynaptic retrograde tracer was used as a control to be able to differentiate between direct and indirect projections. Our tracing experiments showed that the arcuate nucleus of the hypothalamus, as a major site for regulation of food intake, sends only very indirect projections onto the MOB. Indirect projections to MOB also originate from the solitary nucleus which is involved in energy homeostasis. Other indirect projections have been evidenced in areas of the reward circuit such as VTA and accumbens nucleus. In contrast, direct projections to the MOB arise from melanin-concentrating hormone and orexin neurons in the lateral hypothalamus. Functional significances of these projections are discussed in relation to the role of food odors in feeding and reward-related behavior.