Shigehiro Namiki

Reconstructing the Population Activity of Olfactory Output Neurons that Innervate Identifiable Processing Units.

We investigated the functional organization of the moth antennal lobe (AL), the primary olfactory network, by integrating single-cell electrophysiological recording data with geometrical information. The moth AL contains about 60 processing units called glomeruli that are identifiable from one animal to another. We were able to monitor the output information of the AL by recording the activity of a population of output neurons, each of which innervated a single glomerulus. Using compiled in vivo intracellular recordings and staining data from different animals, we mapped the odor-evoked dynamics on a digital atlas of the AL and geometrically reconstructed the population activity. We examined the quantitative relationship between the similarity of olfactory responses and the anatomical distance between glomeruli. Globally, the olfactory response profile was independent of the anatomical distance, although some local features were present. Olfactory response profiles of superficial glomeruli were approximately similar, whereas those of deep glomeruli were different with each other, suggesting network architectures are different in superficial and deep glomerular networks during olfactory processing.

Representation of a mixture of pheromone and host plant odor by antennal lobe projection neurons of the silkmoth Bombyx mori

Pheromone-source orientation behavior can be modified by coexisting plant volatiles. Some host plant volatiles enhance the pheromonal responses of olfactory receptor neurons and increase the sensitivity of orientation behavior in the Lepidoptera species. Although many electrophysiological studies have focused on the pheromonal response of olfactory interneurons, the response to the mixture of pheromone and plant odor is not yet known. Using the silkmoth, Bombyx mori, we investigated the physiology of interneurons in the antennal lobe (AL), the primary olfactory center in the insect brain, in response to a mixture of the primary pheromone component bombykol and cis-3-hexen-1-ol, a mulberry leaf volatile. Application of the mixture enhanced the pheromonal responses of projection neurons innervating the macroglomerular complex in the AL. In contrast, the mixture of pheromone and cis-3-hexen-1-ol had little influence on the responses of projection neurons innervating the ordinary glomeruli whereas other plant odors dynamically modified the response. Together this suggests moths can process plant odor information under conditions of simultaneous exposure to sex pheromone.

Constancy and variability of glomerular organization in the antennal lobe of the silkmoth

We investigated the anatomical organization of glomeruli in the antennal lobes (ALs) of male silkmoths. We reconstructed 10 different ALs and established an identification procedure for individual glomeruli by using size, shape, and position relative to anatomical landmarks. Quantitative analysis of these morphological characteristics supported the validity of our identification strategy. The glomerular organization of the ALs was roughly conserved between different ALs. However, we found individual variations that were reproducibly observed. The combination of a digital atlas with other experimental techniques, such as electrophysiology, optical imaging, and genetics, should facilitate a more in-depth analysis of sensory information processing in silkmoth ALs.

Molecular and neural mechanisms of sex pheromone reception and processing in the silkmoth Bombyx mori

Male moths locate their mates using species-specific sex pheromones emitted by conspecific females. One striking feature of sex pheromone recognition in males is the high degree of specificity and sensitivity at all levels, from the primary sensory processes to behavior. The silkmoth Bombyx mori is an excellent model insect in which to decipher the underlying mechanisms of sex pheromone recognition due to its simple sex pheromone communication system, where a single pheromone component, bombykol, elicits the full sexual behavior of male moths. Various technical advancements that cover all levels of analysis from molecular to behavioral also allow the systematic analysis of pheromone recognition mechanisms. Sex pheromone signals are detected by pheromone receptors expressed in olfactory receptor neurons in the pheromone-sensitive sensilla trichodea on male antennae. The signals are transmitted to the first olfactory processing center, the antennal lobe (AL), and then are processed further in the higher centers (mushroom body and lateral protocerebrum) to elicit orientation behavior toward females. In recent years, significant progress has been made elucidating the molecular mechanisms underlying the detection of sex pheromones. In addition, extensive studies of the AL and higher centers have provided insights into the neural basis of pheromone processing in the silkmoth brain. This review describes these latest advances, and discusses what these advances have revealed about the mechanisms underlying the specific and sensitive recognition of sex pheromones in the silkmoth.

Pheromone responsiveness threshold depends on temporal integration by antennal lobe projection neurons

Significance The olfactory system of male moths exhibits the ability to detect minute amounts of sex pheromones. How this extreme sensitivity is achieved remains unclear. Using optogenetic techniques to activate a pheromone-responsive olfactory receptor neuron, our results reveal that weak olfactory inputs, but not strong inputs, are temporally integrated in second-order projection neurons to promote behavioral responsiveness. Furthermore, temporal integration of strong olfactory inputs is inhibited by GABAergic mechanisms, indicating that GABA signaling suppresses the amplification of strong stimuli. The timescale of this temporal integration corresponds well to the temporal dynamics of odor signals in the natural environment, suggesting that the olfactory systems of male moths use this mechanism to detect weak pheromone signals in the air. The olfactory system of male moths has an extreme sensitivity with the capability to detect and recognize conspecific pheromones dispersed and greatly diluted in the air. Just 170 molecules of the silkmoth (Bombyx mori) sex pheromone bombykol are sufficient to induce sexual behavior in the male. However, it is still unclear how the sensitivity of olfactory receptor neurons (ORNs) is relayed through the brain to generate high behavioral responsiveness. Here, we show that ORN activity that is subthreshold in terms of behavior can be amplified to suprathreshold levels by temporal integration in antennal lobe projection neurons (PNs) if occurring within a specific time window. To control ORN inputs with high temporal resolution, channelrhodopsin-2 was genetically introduced into bombykol-responsive ORNs. Temporal integration in PNs was only observed for weak inputs, but not for strong inputs. Pharmacological dissection revealed that GABAergic mechanisms inhibit temporal integration of strong inputs, showing that GABA signaling regulates PN responses in a stimulus-dependent fashion. Our results show that boosting of the PNs’ responses by temporal integration of olfactory information occurs specifically near the behavioral threshold, effectively defining the lower bound for behavioral responsiveness.

Information flow through neural circuits for pheromone orientation

Moths use a sophisticated olfactory navigation strategy for resource localization. Here we investigate the neuronal circuits involved in sensory processing to generate locomotor commands for pheromone-source orientation in the moth. We identify a candidate pathway for pheromone processing in the protocerebrum using a mass-staining technique. Our intracellular recordings of pheromone responsiveness detect four major circuits, including a newly identified unstructured neuropil, the superior medial protocerebrum, which supplies output to the lateral accessory lobe (LAL), the premotor centre for walking commands. Interneurons innervating the lower division of the LAL elicited longer responses than those innervating the upper division. Descending interneurons innervating the lower division of the LAL showed a state-dependent flip-flop response. In contrast, input from other visual areas in the protocerebrum mostly converge onto the upper division of the LAL. These results reveal the basic organization of the LAL: the upper division is identified as a protocerebral hub that receives inputs from various areas, while the lower division generates long-lasting activity for locomotor command.

Use of bilateral information to determine the walking direction during orientation to a pheromone source in the silkmoth Bombyx mori

Odor source localization is an important animal behavior. Male moths locate mates by tracking sex pheromone emitted by conspecific females. During this type of behavior, males exhibit a combination of upwind surge and zigzagging flight. Similarly, the male walking moth Bombyx mori responds to transient pheromone exposure with a surge in movement, followed by sustained zigzagging walking. The initial surge direction is known to be influenced by the pheromone input pattern. Here, we identified the sensory input patterns that determine the initial walking direction of males. We first quantified the stimulus by measuring electroantennogram values, which were used as a reference for subsequent tests. We used a brief stimulus pulse to examine the relationship between sensory stimulus patterns and the turning direction of initial surge. We found that the difference in input timing and intensity between left and right antennae affected the walking direction, indicating that B. mori integrate bilateral pheromone information during orientation behavior. When we tested pheromone stimulation for longer periods, turning behavior was suppressed, which was induced by stimulus cessation. This study contributes toward understanding efficient strategies for odor-source localization that is utilized by walking insects.

Heterogeneity in dendritic morphology of moth antennal lobe projection neurons

A population of projection neurons (PNs) in the antennal lobe (AL) integrates sensory information from the antenna and is essential for processing odor information in the insect brain. We examined the anatomy of this neuronal population in the brain of the silkmoth Bombyx mori. Using intracellular dye injection, we labeled a total of 246 PNs and systematically analyzed their morphological features, including the soma position, antennocerebral tract, and number of innervating glomeruli. For example, we analyzed PNs that had somata in the different cell clusters, innervated overlapping but different groups of glomeruli, and ran through different pathways. We also identified glomeruli innervated by PNs using a previously established procedure that first classifies glomeruli into regional groups and then identifies individual glomeruli. We analyzed uniglomerular PNs (75.6% of the total) and found heterogeneity in the dendritic morphology of the PNs that was dependent on the regions and/or the innervating glomeruli. For example, most PNs innervating the macroglomerular complex did not have extraglomerular processes, whereas most PNs innervating ordinary glomeruli did. Moreover, PNs innervating the toroid glomerulus showed heterogeneity in their dendritic morphology. These PNs had dendritic arborization in different areas within the glomerulus. We found that, in some cases, the innervation pattern of the PN dendrite correlated with individual variation in the glomerular organization. These results indicate that PNs are not homogeneous populations, and in some cases morphological heterogeneity in PNs correlated with change in glomerular organization in the silkmoth AL. J. Comp. Neurol. 519:3367–3386, 2011.

Sex-linked transcription factor involved in a shift of sex-pheromone preference in the silkmoth Bombyx mori.

In the sex-pheromone communication systems of moths, odorant receptor (Or) specificity as well as higher olfactory information processing in males should be finely tuned to the pheromone of conspecific females. Accordingly, male sex-pheromone preference should have diversified along with the diversification of female sex pheromones; however, the genetic mechanisms that facilitated the diversification of male preference are not well understood. Here, we explored the mechanisms involved in a drastic shift in sex-pheromone preference in the silkmoth Bombyx mori using spli mutants in which the genomic structure of the gene Bmacj6, which encodes a class IV POU domain transcription factor, is disrupted or its expression is repressed. B. mori females secrete an ∼11:1 mixture of bombykol and bombykal. Bombykol alone elicits full male courtship behavior, whereas bombykal alone shows no apparent activity. In the spli mutants, the behavioral responsiveness of males to bombykol was markedly reduced, whereas bombykal alone evoked full courtship behavior. The reduced response of spli males to bombykol was explained by the paucity of bombykol receptors on the male antennae. It was also found that, in the spli males, neurons projecting into the toroid, a compartment in the brain where bombykol receptor neurons normally project, responded strongly to bombykal. The present study highlights a POU domain transcription factor, Bmacj6, which may have caused a shift of sex-pheromone preference in B. mori through Or gene choice and/or axon targeting.

Layer III Neurons Control Synchronized Waves in the Immature Cerebral Cortex

Correlated spiking activity prevails in immature cortical networks and is believed to contribute to neuronal circuit maturation; however, its spatiotemporal organization is not fully understood. Using wide-field calcium imaging from acute whole-brain slices of rat pups on postnatal days 1–6, we found that correlated spikes were initiated in the anterior part of the lateral entorhinal cortex and propagated anteriorly to the frontal cortex and posteriorly to the medial entorhinal cortex, forming traveling waves that engaged almost the entire cortex. The waves were blocked by ionotropic glutamatergic receptor antagonists but not by GABAergic receptor antagonists. During wave events, glutamatergic and GABAergic synaptic inputs were balanced and induced UP state-like depolarization. Magnified monitoring with cellular resolution revealed that the layer III neurons were first activated when the waves were initiated. Consistent with this finding, layer III contained a larger number of neurons that were autonomously active, even under a blockade of synaptic transmission. During wave propagation, the layer III neurons constituted a leading front of the wave. The waves did not enter the parasubiculum; however, in some cases, they were reflected at the parasubicular border and propagated back in the opposite direction. During this reflection process, the layer III neurons in the medial entorhinal cortex maintained persistent activity. Thus, our data emphasize the role of layer III in early network behaviors and provide insight into the circuit mechanisms through which cerebral cortical networks maturate.