Hitoshi Aonuma

Pheromone processing center in the protocerebrum of Bombyx mori revealed by nitric oxide-induced anti-cGMP immunocytochemistry.

The antennal lobe (AL) of the male silkworm moth Bombyx mori contains 60 ± 2 ventrally located antennal glomeruli and a dorsal macroglomerular complex (MGC) consisting of three subdivisions. The response patterns of MGC projection neurons (PNs) to pheromonal stimuli correlate with their dendritic arborization in the subdivisions of the MGC. However, the representation of this pheromonal information in the lateral protocerebrum (LPC), which is the target site of the AL PNs, is not well known. We performed nitric oxide (NO)‐induced anti‐cGMP immunohistochemistry and found that the PNs which respond to the major pheromone component (bombykol) express strong immunoreactivity. They project to a specific area, the delta area in the inferior lateral protocerebrum (ΔILPC), which clearly represents the processing center for the major pheromone component. Furthermore, to examine the projection sites in the LPC from each subdivision of the MGC, we performed double‐labeling of each type of MGC‐PNs, combined with NO‐induced anti‐cGMP immunohistochemistry. We revealed that projections from each subdivision of the MGC overlapped or separated in specific regions of the ΔILPC. These results suggest that integration and segregation of pheromone information may occur in the ΔILPC. J. Comp. Neurol. 481:340–351, 2005.

Discrimination of Conspecific Individuals via Cuticular Pheromones by Males of the Cricket Gryllus bimaculatus

Abstract Cuticular substances on the body surface of crickets serve as pheromones that elicit a variety of different behaviors in male crickets. Antennal contact between males and females resulted in courtship behavior, and that between two males resulted in aggressive displays. As a first step in elucidating how crickets recognize and discriminate individuals, behavioral responses of male individuals to cuticular substances of conspecific males or females were investigated. The behavioral responses of males to antennal or palpal stimulation with an isolated antenna from a male or a female were recorded. To both antennal and palpal stimulation with female antennae, the majority of males responded with courtship behavior; to stimulation with male antennae, males responded with aggressive displays. To gain insight into the chemical nature of the behaviorally relevant components, isolated antennae were washed in either n-hexane, acetone or ethanol before behavior assays. Washed antennae no longer elicited courtship or aggressive responses in males. Next, polypropylene fibers were smeared with substances from the body surface of females and used for antennal stimulation. This experiment showed that the quality and quantity of cuticular substances appear to be highly age-dependent. Significantly more males responded with courtship behavior to cuticular substances from younger females. Isolated males generally showed higher levels of aggression than males reared in groups. Grouped males also were more likely to display courtship behavior towards antennae from younger females, and aggressive behavior towards antennae from older females. These results suggest that male discrimination of mating partners depends on the nature of female cuticular substances.

The Nitric Oxide/Cyclic GMP Pathway in the Olfactory Processing System of the Terrestrial Slug Limax marginatus

Abstract To examine the distribution of nitric oxide (NO)-generative cells and NO-responsive cells in the tentacles and procerebral lobes (olfactory processing center) of terrestrial slugs, we applied NADPH diaphorase (NADPH-d) histochemistry and NO-induced cyclic GMP (cGMP)-like immunohistochemistry. We found that NADPH-d reactive cells/fibers and cGMP-like immunoreactive cells/fibers were different, but they were localized adjacent to each other, in both the tentacles and the procerebral lobes. Then, we measured the concentration of NO that was generated around the procerebral lobes using an NO sensitive electrode, when the olfactory nerve was electrically stimulated as a replacement for an odorant stimulus. Stimulation of the olfactory nerve evoked an increase in NO concentration at nanomolar levels, suggesting that binding of nanomolar concentrations of NO to the prosthetic heme group activates soluble guanylyl cyclase. Taken together with previously reported physiological data, our results, therefore, showed that the NO/cGMP pathways are involved in slug olfactory processing.

Social interactions influence dopamine and octopamine homeostasis in the brain of the ant Formica japonica.

SUMMARY In ants, including Formica japonica, trophallaxis and grooming are typical social behaviors shared among nestmates. After depriving ants of either food or nestmates and then providing them with either food or nestmates, a behavioral change in type and frequency of social interactions was observed. We hypothesized that starvation and isolation affected levels of brain biogenic amines including dopamine (DA) and octopamine (OA) – neuromediators modifying various insect behaviors – and tested the relationship between brain biogenic amines and social behaviors of stressed ants. Ants starved for 7 days contained lower brain DA levels and they did not perform trophallaxis toward nestmates. Feeding starved ants sucrose solution re-established trophallaxis but not brain DA levels. The performance of trophallaxis induced recovery of brain DA content to the level of untreated ants. Ants that were isolated for 2 days displayed markedly increased OA levels, which following nestmate interactions, returned to levels similar to those of control (non-isolated) ants and ants isolated for 1 h. We conclude that: (1) starvation reduced brain DA level but had no significant effect on brain OA (trophallaxis recovered the brain DA levels), and (2) isolation increased brain OA level but had no effect on brain DA (trophallaxis and grooming events recovered the brain OA levels). We suggest that social interactions with nestmates influenced brain biogenic amine homeostasis in stressed F. japonica.

De Novo synthesis of CREB in a presynaptic neuron is required for synaptic enhancement involved in memory consolidation

Interaction between the activator type of cyclic AMP response element binding protein (CREB1) and the repressor type (CREB2) results in determining the emergence of long‐lasting synaptic enhancement involved in memory consolidation. However, we still do not know whether the constitutively expressed forms of CREB are enough or the newly synthesized forms are required for the synaptic enhancement. In addition, if the newly synthesized forms are needed, we must determine the time for translation of CREB from its mRNA. We applied the methods of RNA interference and real‐time polymerase chain reaction (PCR) to CREB in the cerebral giant cells of Lymnaea. The cerebral giant cells play an important role in associative learning and employ a CREB cascade for the synaptic enhancement to neurons such as the B1 motoneurons. We injected the small interfering RNA (siRNA) of CREB1 or CREB2 into the cerebral giant cells and examined the changes in amplitude of excitatory postsynaptic potential (EPSP) recorded in the B1 motoneurons. The changes in the amounts of CREB1 and CREB2 mRNAs were also examined in the cerebral giant cells. The EPSP amplitude was suppressed 15 min after injection of CREB1 siRNA, whereas that was augmented 60 min after injection of CREB2 siRNA. In the latter case, the decrease in the amount of CREB2 mRNA was confirmed by real‐time PCR. Our results showed that the de novo synthesized forms of CREB are required within tens of minutes for the synaptic enhancement in memory consolidation.

Effects of Previous Experience on the Agonistic Behaviour of Male Crickets, Gryllus bimaculatus

Abstract Male solitary animals frequently enter aggressive interactions with conspecific individuals to protect their territory or to gain access to females. After an agonistic encounter, the loser (subordinate individual) changes its behaviour from aggression to avoidance. We investigated agonistic interactions between pairs of male crickets to understand how dominance is established and maintained. Two naïve males readily entered into agonistic interactions. Fights escalated in a stereotyped manner and were concluded with the establishment of dominance. If individuals were isolated after the first encounter and placed together 15 minutes later, subordinate crickets tended to avoid any further contact with the former dominant opponent. Moreover, subordinate males also avoided unfamiliar dominant and naïve opponents. They displayed aggressive behaviour only towards unfamiliar subordinate opponents. This suggests that the subordinate male change their behaviour depending on the dominance status of the opponent. Dominant crickets, in contrast, displayed aggressive behaviour towards familiar as well as unfamiliar opponents. If the interval between the first and second encounter was longer than 30 minutes, the former subordinate male showed aggressive behaviour again. However, if the subordinate cricket was paired with the same opponent three consecutive times within 45 minutes, it avoided the former dominant opponent for up to 6 hours following the third encounter. Our results suggest that the maintenance of dominance in male crickets depends largely on the behavioural change of subordinate individuals. Possible mechanisms to maintain dominance are discussed.

Identification and expression analysis of the genes involved in serotonin biosynthesis and transduction in the field cricket Gryllus bimaculatus

Serotonin (5‐HT) modulates various aspects of behaviours such as aggressive behaviour and circadian behaviour in the cricket. To elucidate the molecular basis of the cricket 5‐HT system, we identified 5‐HT‐related genes in the field cricket Gryllus bimaculatus DeGeer. Complementary DNA of tryptophan hydroxylase and phenylalanine‐tryptophan hydroxylase, which convert tryptophan into 5‐hydroxy‐L‐tryptophan (5‐HTP), and that of aromatic L‐amino acid decarboxylase, which converts 5‐HTP into 5‐HT, were isolated from a cricket brain cDNA library. In addition, four 5‐HT receptor genes (5‐HT1A, 5‐HT1B, 5‐HT2α, and 5‐HT7) were identified. Expression analysis of the tryptophan hydroxylase gene TRH and phenylalanine‐tryptophan hydroxylase gene TPH, which are selectively involved in neuronal and peripheral 5‐HT synthesis in Drosophila, suggested that two 5‐HT synthesis pathways co‐exist in the cricket neuronal tissues. The four 5‐HT receptor genes were expressed in various tissues at differential expression levels, suggesting that the 5‐HT system is widely distributed in the cricket.

Distribution of NADPH-diaphorase-positive ascending interneurones in the crayfish terminal abdominal ganglion

Abstract. Previous neuropharmacological studies have described the presence of a nitric oxide-cGMP signalling pathway in the crayfish abdominal nervous system. In this study we have analysed the distribution of putative nitric oxide synthase (NOS)-containing ascending interneurones in the crayfish terminal abdominal ganglion using NADPH-diaphorase (NADPHd) histochemistry. Ascending intersegmental interneurones were stained intracellularly using the fluorescent dye Lucifer yellow and the ganglia containing the stained interneurones subsequently processed for NADPHd activity. Fluorescence persisted throughout histochemical processing. These double-labelling experiments showed that 12 of 18 identified ascending interneurones were NADPHd positive. Thus many ascending interneurones that process mechanosensory signals in the terminal ganglion may contain NOS, and are themselves likely sources of NO which is known to modulate their synaptic inputs. Three clear relationships emerged from our analysis between the effects of NO on the synaptic inputs of interneurones, their output properties and their staining for NADPH-diaphorase. First were class 1 interneurones with no local outputs in the terminal ganglion, the NE type interneurones, which had sensory inputs that were enhanced by NO and were NADPHd positive. Second were class 1 interneurones with local and intersegmental output effects that had sensory inputs that were also enhanced by NO but were NADPHd negative. Third were class 2 interneurones with local and intersegmental outputs that had synaptic inputs that were depressed by the action of NO but were NADPHd positive. These results suggest that NO could selectively enhance specific synaptic connections and sensory processing pathways in local circuits.

Distribution of NO-Induced cGMP-Like Immunoreactive Neurones in the Abdominal Nervous System of the Crayfish, Procambarus clarkii

Abstract Nitric oxide (NO) acts as a signalling molecule by activating soluble guanylate cyclase and causing accumulation of the second messenger cyclic guanosine 3′,5′-monophosphate (cGMP) in target cells. In order to detect the presence of NO-cGMP signalling pathway in the crayfish abdominal nervous system, accumulation of NO-induced cGMP was investigated by anti-cGMP immunochemistry. Some preparations were incubated in a high-K+ saline containing an inhibitor of cGMP-degrading phosphodiesterase, 3-isobutyl-1-methyxanthine (IBMX), to activate NO generating neurones, which could release NO in the ganglion, and then immunohistochemistry using an anti-cGMP antibody was performed. The other preparations were incubated in NO donor, sodium nitroprusside (SNP) saline containing IBMX before anti-cGMP immunohistochemistry was performed. The distribution of cGMP-like immunoreactive neurones in high-K+ treated preparations was similar to that of cGMP-like immunoreactive neurones in NO donor treated preparations. About 70-80 cell bodies and many neuronal branches in the neuropilar area of the ganglion were stained, although no neurones showed immunoreactivity unless preparations were activated by either high-K+ or the NO donor. Some of them were identical neurones, and they were intersegmental ascending interneurones and motor neurones. Sensory afferents that innervates hind gut showed strong cGMP-like immunoreactivity, although no mechanosensory afferents showed any immunoreactivity. These results strongly suggest the presence of an NO-cGMP signalling pathway that regulates neuronal events in the abdominal nervous system of the crayfish.

Distribution of GABAergic premotor nonspiking local interneurones in the terminal abdominal ganglion of the crayfish

The inhibitory neurotransmitter of premotor nonspiking local interneurones in the crayfish terminal abdominal ganglion was investigated physiologically and immunocytochemically. Depolarization of a nonspiking interneurone evoked a hyperpolarization in a uropod motor neurone. The amplitude of hyperpolarization in the motor neurone was gradually decreased under low‐calcium/high‐magnesium saline. Local pressure injection of γ‐aminobutyric acid (GABA) into the neuropil caused a similar hyperpolarization of the motor neurone. These physiological studies suggested a GABAergic inhibitory interaction between nonspiking interneurones and the motor neurones. Premotor nonspiking interneurones are classified into two subgroups of posterolateral (PL) and anterolateral (AL) interneurones, and AL interneurones are further divided into three subtypes. A combination of intracellular staining from nonspiking local interneurones with Lucifer yellow and immunocytochemical staining with an antiserum directed against GABA revealed that all the PL interneurones sampled in this study showed GABA‐like immunoreactivity. A population of cell bodies (n = 6–11) with a small diameter (15–30 μm) packed together forming a cluster showed GABA‐like immunoreactivity, and the cell bodies of most PL interneurones were found in this cluster. To compare the number and the pattern of main branches of PL interneurones, cells were classified into three identifiable sets of interneurones, called PL‐1, PL‐2, and PL‐3. By contrast, about one‐half of AL interneurones, especially the third subtype of AL interneurones, which have cell bodies located ventrolaterally in the ganglion, did not show GABA‐like immunoreactivity. Furthermore, the position of cell bodies of GABA‐immunoreactive AL interneurones was scattered compared to that of PL interneurones. J. Comp. Neurol. 389: 139–148,1997.