Hiroyuki Ai

Topographic organization of sensory afferents of Johnston's organ in the honeybee brain.

Johnstons organ (JO) in insects is a multicellular mechanosensory organ stimulated by movement of the distal part of the antenna. In honeybees JO is thought to be a primary sensor detecting air‐particle movements caused by the waggling dance of conspecifics. In this study projection patterns of JO afferents within the brain were investigated. About 720 somata, distributed around the periphery of the second segment of the antenna (pedicel), were divided into three subgroups based on their soma location: an anterior group, a ventral group, and a dorsal group. These groups sent axons to different branches (N2 to N4) diverged from the antennal nerve. Dye injection into individual nerve branches revealed that all three groups of afferents, having fine collaterals in the dorsal lobe, sent axons broadly through tracts T6I, T6II, and T6III to terminate ipsilaterally in the medial posterior protocerebral lobe, the dorsal region of the subesophageal ganglion, and the central posterior protocerebral lobe, respectively. Within these termination fields only axon terminals running in T6I were characterized by thick processes with large varicosities. Differential staining using fluorescent dyes revealed that the axon terminals of the three groups were spatially segregated, especially in T6I, showing some degree of somatotopy. This spatial segregation was not observed in axon terminals running in other tracts. Our results show that projection patterns of JO afferents in the honeybee brain fundamentally resemble those in the dipteran brain. The possible roles of extensive termination fields of JO afferents in parallel processings of mechanosensory signals are discussed. J. Comp. Neurol. 502:1030–1046, 2007.

Modular organization of the silkmoth antennal lobe macroglomerular complex revealed by voltage-sensitive dye imaging.

SUMMARY We succeeded in clarifying the functional synaptic organization of the macroglomerular complex (MGC) of the male silkmoth Bombyx mori by optical recording with a voltage-sensitive dye. Sensory neurons in the antennae send their axons down either the medial nerve (MN) or lateral nerve (LN), depending on whether they are located on the medial or lateral flagella. Pheromone-sensitive fibers in the MN are biased towards the medial MGC, and those in the LN are biased towards the lateral MGC in the antennal lobe. In our optical recording experiments, the postsynaptic activities in the MGC were characterized by pharmacological analysis. Postsynaptic activities in the MGC were separated from sensory activities under Ca2+-free conditions, and subsequently the inhibitory postsynaptic activities were separated by applying bicuculline. We found that the inhibitory postsynaptic responses always preceded the postsynaptic responses separated under Ca2+-free conditions. Moreover, the excitatory postsynaptic activities were calculated by subtracting the inhibitory potentials from the posysynaptic activities separated under Ca2+-free conditions. When the MN was stimulated, the amplitudes of the excitatory postsynaptic activities in the central toroid, the medial toroid and the medial cumulus were selectively higher than those in the other areas. By contrast, when the LN was stimulated, excitatory postsynaptic activities were evoked in areas in both the lateral toroid and the lateral cumulus. The inhibitory postsynaptic activities were equally distributed throughout the whole MGC. These data suggest that there is a modular organization to the MGC such that information from the two main branches of the antenna is segregated to different sub-regions of the MGC glomeruli.

Response characteristics of vibration-sensitive interneurons related to Johnston's organ in the honeybee, Apis mellifera.

Honeybees detect airborne vibration by means of Johnstons organ (JO), located in the pedicel of each antenna. In this study we identified two types of vibration‐sensitive interneurons with arborizations in the primary sensory area of the JO, namely, the dorsal lobe‐interneuron 1 (DL‐Int‐1) and dorsal lobe‐interneuron 2 (DL‐Int‐2) using intracellular recordings combined with intracellular staining. For visualizing overlapping areas between the JO sensory terminals and the branches of these identified interneurons, the three‐dimensional images of the individual neurons were registered into the standard atlas of the honeybee brain (Brandt et al. [2005] J Comp Neurol 492:1–19). Both DL‐Int‐1 and DL‐Int‐2 overlapped with the central terminal area of receptor neurons of the JO in the DL. For DL‐Int‐1 an on–off phasic excitation was elicited by vibrational stimuli applied to the JO when the spontaneous spike frequency was low, whereas tonic inhibition was induced when it was high. Moreover, current injection into a DL‐Int‐1 led to changes of the response pattern from on–off phasic excitation to tonic inhibition, in response to the vibratory stimulation. Although the vibration usually induced on–off phasic excitation in DL‐Int‐1, vibration applied immediately after odor stimulation induced tonic inhibition in it. DL‐Int‐2 responded to vibration stimuli applied to the JO by a tonic burst and were most sensitive to 265 Hz vibration, which is coincident with the strongest frequency of airborne vibrations arising during the waggle dance. These results suggest that DL‐Int‐1 and DL‐Int‐2 are related to coding of the duration of the vibration as sensed by the JO. J. Comp. Neurol. 515:145–160, 2009.

Neural pathways for cardiac reflexes triggered by external mechanical stimuli in larvae of Bombyx mori

Abstract Stimuli applied to mechano-exteroceptors induce the following five types of reflex cardiac responses in larvae of Bombyx mori. (1) Tactile stimuli applied to sensillar setae of the anal proleg induce antidromic heartbeat. (2) The stimuli simultaneously inhibit orthodromic heartbeat. These stimuli increase the heart rate if applied during a phase of antidromic heartbeat. The visceral nerve arises from the frontal ganglion. The anterior cardiac nerves branching out of the visceral nerve were found to trigger antidromic heartbeat. Posterior cardiac nerves branching out of the visceral nerve are the inhibitory motor nerves which inhibit orthodromic heartbeat. These posterior cardiac nerves also contain motor nerves for alary muscles in the 2nd abdominal segment. (3) Contraction of the alary muscle increases heart tone and results in acceleration of antidromic heartbeat. (4) Tactile stimuli to the antennae accelerate orthodromic heartbeat. The motor pathway is through the paired dorsal nerves of the 1st abdominal ganglion in a segment which lacks a pair of alary muscles. (5) Contraction of the alary muscles in the 7th and 8th abdominal segments evoked by these stimuli contributes to acceleration of the orthodromic heartbeat. The motor pathways for this are in the paired dorsal nerves of the 7th abdominal ganglion.

Biogenic amines evoke heartbeat reversal in larvae of the sweet potato hornworm, Agrius convolvuli.

The sweet potato hornworm, Agrius convolvuli, possesses a pair of anterior cardiac nerves innervating the dorsal vessel. The anterior cardiac nerves branch off the visceral nerve that arises posteriorly from the frontal ganglion. Heartbeat reversal from anterograde heartbeat to posterograde heartbeat is triggered by the anterior cardiac nerves. Application of octopamine (OA) during the anterograde heartbeat phase reverses the anterograde heartbeat to the posterograde heartbeat, while application of OA during the phase of posterograde heartbeat accelerates heartbeat. The heartbeat reversal from anterograde heartbeat to posterograde heartbeat evoked by stimuli applied to the visceral nerve is blocked by application of the octopaminergic antagonists, phentolamine and chlorpromazine. The results suggest that OA may be a neurotransmitter for the anterior cardiac nerve. The alary muscle of the second segment receives excitatory innervation from the posterior cardiac nerve and from the nerve which extends from the second abdominal ganglion. Activation of the alary muscle results in acceleration of posterograde heartbeat. Other neurotransmitters, besides OA, may take part in the resultant acceleration.

Vibration receptive sensilla on the wing margins of the silkworm moth Bombyx mori.

Bristles along the wing margins (wm-bristles) of the silkworm moth, Bombyx mori, were studied morphologically and electrophysiologically. The male moth has ca. 50 wm-bristles on each forewing and hindwing. Scanning electron microscopy revealed that these wm-bristles are typical mechanosensilla. Leuco-methylene blue staining demonstrated that each wm-bristle has a single receptor neuron, which is also characteristic of the mechanosensillum. The receptor neuron responded to vibrating air currents but did not respond to a constant air current. The wm-bristles showed clear directional sensitivity to vibrating air currents. The wm-bristles were classified into two types, type I and type II, by their response patterns to sinusoidal movements of the bristle. The neuron in type I discharged bursting spikes immediately following stimulation onset and also discharged a single spike for each sinusoidal cycle for frequencies less than ca. 60 Hz. The neuron in type II only responded to vibrations over 40 Hz and, specifically at 75 Hz, discharged a single spike for each sinusoidal cycle throughout the stimulation period. These results suggest that the two types of wm-bristles are highly tuned in different ways to detect vibrations due to the wing beat. The roles of the wm-bristles in the wing beat are discussed.

Cardiac reflexes and their neural pathways in lepidopterous insects

Abstract Previously described salines for lepidoptera did not maintain a constant heart rate for a very long. We have been successful in maintaining a normal heartbeat for many hours in a newly designed saline. This saline was also suitable for maintaining normal neuromuscular junctional potentials. The cardiac reflexes studied in larvae of Bombyx and Agrius were five types of cardiac responses induced by mechanical stimuli to sensillar setae. The cardiac responses were caused by electrical stimulation of nerves in the reflex pathways. The antidromic heartbeat was triggered even in larvae before the 5th instar by stimulation of axons in the visceral nerve arising from the frontal ganglion and terminating at the aorta, while spontaneous heartbeat reversal started to occur in wandering larvae. Other axons in the visceral nerve terminate at the rear end of the heart. Electrical stimuli to the nerves caused cardiac inhibition of the orthodromic heartbeat. Nerves extending from the visceral nerve to the alary muscles of the 2nd abdominal segment contain axons to increase the tone of the muscles. Nerves extending from the 7th abdominal ganglion to the most posterior alary muscles also contain axons to increase the tone of the muscles, and were responsible for acceleration of the antidromic and orthodromic heartbeat, respectively.

Vibration-Processing Interneurons in the Honeybee Brain

The afferents of the Johnstons organ (JO) in the honeybee brain send their axons to three distinct areas, the dorsal lobe, the dorsal subesophageal ganglion (DL-dSEG), and the posterior protocerebral lobe (PPL), suggesting that vibratory signals detected by the JO are processed differentially in these primary sensory centers. The morphological and physiological characteristics of interneurons arborizing in these areas were studied by intracellular recording and staining. DL-Int-1 and DL-Int-2 have dense arborizations in the DL-dSEG and respond to vibratory stimulation applied to the JO in either tonic excitatory, on-off-phasic excitatory, or tonic inhibitory patterns. PPL-D-1 has dense arborizations in the PPL, sends axons into the ventral nerve cord (VNC), and responds to vibratory stimulation and olfactory stimulation simultaneously applied to the antennae in long-lasting excitatory pattern. These results show that there are at least two parallel pathways for vibration processing through the DL-dSEG and the PPL. In this study, Honeybee Standard Brain was used as the common reference, and the morphology of two types of interneurons (DL-Int-1 and DL-Int-2) and JO afferents was merged into the standard brain based on the boundary of several neuropiles, greatly supporting the understanding of the spatial relationship between these identified neurons and JO afferents. The visualization of the region where the JO afferents are closely appositioned to these DL interneurons demonstrated the difference in putative synaptic regions between the JO afferents and these DL interneurons (DL-Int-1 and DL-Int-2) in the DL. The neural circuits related to the vibration-processing interneurons are discussed.

Spatio-temporal activities in the antennal lobe analyzed by an optical recording method in the male silkworm moth Bombyx mori

Optical recordings with a voltage-sensitive dye showed that the spatio-temporal pattern of depolarizing responses evoked by electrical stimulation of antennal nerve (AN) was non-homologously distributed in the antennal lobe (AL) of the male silkworm moth, Bombyx mori. Time courses of postsynaptic activities and GABAergic inhibitory potentials of AL neurons were individually demonstrated by pharmacological experiments, i.e. Ca2+ free and bicuculline conditions. GABAergic inhibitory potentials began with a ca. 3 ms delay from the beginning of the postsynaptic activities. Intensity of the postsynaptic activities and GABAergic inhibitory potentials were non-homologously distributed in the AL. Relatively strong postsynaptic activities and GABAergic inhibitory potentials were consistently observed in some parts of the macroglomerular complex (MGC) and/or in some ordinary glomeruli (Gs) in the medial and ventral part of the AL.

Morphological analysis of the primary center receiving spatial information transferred by the waggle dance of honeybees.

The waggle dancers of honeybees encodes roughly the distance and direction to the food source as the duration of the waggle phase and the body angle during the waggle phase. It is believed that hive‐mates detect airborne vibrations produced during the waggle phase to acquire distance information and simultaneously detect the body axis during the waggle phase to acquire direction information. It has been further proposed that the orientation of the body axis on the vertical comb is detected by neck hairs (NHs) on the prosternal organ. The afferents of the NHs project into the prothoracic and mesothoracic ganglia and the dorsal subesophageal ganglion (dSEG). This study demonstrates somatotopic organization within the dSEG of the central projections of the mechanosensory neurons of the NHs. The terminals of the NH afferents in dSEG are in close apposition to those of Johnstons organ (JO) afferents. The sensory axons of both terminate in a region posterior to the crossing of the ventral intermediate tract (VIT) and the maxillary dorsal commissures I and III (MxDCI, III) in the subesophageal ganglion. These features of the terminal areas of the NH and JO afferents are common to the worker, drone, and queen castes of honeybees. Analysis of the spatial relationship between the NH neurons and the morphologically and physiologically characterized vibration‐sensitive interneurons DL‐Int‐1 and DL‐Int‐2 demonstrated that several branches of DL‐Int‐1 are in close proximity to the central projection of the mechanosensory neurons of the NHs in the dSEG. J. Comp. Neurol. 521:2570–2584, 2013.