Kouji Yasuyama

Localization of choline acetyltransferase-expressing neurons in Drosophila nervous system

A variety of approaches have been developed to localize neurons and neural elements in nervous system tissues that make and use acetylcholine (ACh) as a neurotransmitter. Choline acetyltransferase (ChAT) is the enzyme catalyzing the biosynthesis of ACh and is considered to be an excellent phenotypic marker for cholinergic neurons. We have surveyed the distribution of choline acetyltransferase (ChAT)‐expressing neurons in the Drosophila nervous system detected by three different but complementary techniques. Immunocytochemistry, using anti‐ChAT monoclonal antibodies results in identification of neuronal processes and a few types of cell somata that contain ChAT protein. In situ hybridization using cRNA probes to ChAT messenger RNA results in identification of cell bodies transcribing the ChAT gene. X‐gal staining and/or β‐galactosidase immunocytochemistry of transformed animals carrying a fusion gene composed of the regulatory DNA from the ChAT gene controlling expression of a lacZ reporter has also been useful in identifying cholinergic neurons and neural elements. The combination of these three techniques has revealed that cholinergic neurons are widespread in both the peripheral and central nervous system of this model genetic organism at all but the earliest developmental stages. Expression of ChAT is detected in a variety of peripheral sensory neurons, and in the brain neurons associated with the visual and olfactory system, as well as in neurons with unknown functions in the cortices of brain and ganglia. Microsc. Res. Tech. 45:65–79, 1999.

Extraretinal Photoreceptors at the Compound Eye's Posterior Margin in Drosophila melanogaster

Many invertebrates have supplementary extraocular photoreceptors that often are implicated in circadian rhythms. An extraretinal group of candidate photoreceptors in the fruit fly, Drosophila melanogaster, has been revealed previously at the posterior margin of the compound eye by using a photoreceptor‐specific monoclonal antibody (Hofbauer and Buchner [1989] Naturwissen 76:335–336), but it never has been characterized. Here, we report the fine structure of this cell cluster reported by Hofbauer and Buchner, which is called “eyelet,” as well as the further candidacy of their visual pigment and neurotransmitter. Eyelet forms a specialized, pigmented organ with cells that have numerous microvilli arranged into coherent rhabdomeres. The presence of rhabdomeric microvilli is a defining feature of a photoreceptor, reported here for the first time in eyelet. The rhabdomeres exhibit Rh6 opsin‐like immunoreactivity, which provides evidence that the photoreceptors are functional: they fail to immunostain with antibodies against NINAE (Rh1), Rh4, or Rh5. The photoreceptors have been shown previously to exhibit histamine‐like immunoreactivity, but they also stain with a monoclonal antiserum raised against Drosophila choline acetyltransferase (ChAT), suggesting that the photoreceptors not only may contain histamine but also can synthesize acetylcholine. A ChAT‐immunoreactive axon bundle originating from eyelet terminates in the cortex of the anterior medulla. This bundle also is seen with reduced silver stains. Electron microscopic examination revealed four axon profiles of similar size in this bundle, indicating that eyelet contains at least four photoreceptors. The pathway of eyelets axon bundle coincides with the precocious pathway of Bolwigs nerve that arises from the larval organ of sight. The origin and possible function of eyelet are discussed. J. Comp. Neurol. 412:193–202, 1999.

Synaptic connections of PDF‐immunoreactive lateral neurons projecting to the dorsal protocerebrum of Drosophila melanogaster

Recent studies in Drosophila melanogaster indicate that the neuropeptide pigment‐dispersing factor (PDF) is an important output signal from a set of major clock neurons, s‐LNvs (small ventral lateral neurons), which transmit the circadian phase to subsets of other clock neurons, DNs (dorsal neurons). Both s‐LNvs and DNs have fiber projections to the dorsal protocerebrum of the brain, so that this area is a conspicuous locus for coupling between different subsets of clock neurons. To unravel the neural circuits underlying the flys circadian rhythms, we examined the detailed subcellular morphology of the PDF‐positive fibers of the s‐LNvs in the dorsal protocerebrum, focusing on their synaptic connections, using preembedding immunoelectron microscopy. To examine the distribution of synapses, we also reconstructed the three‐dimensional morphology of PDF‐positive varicosities from fiber profiles in the dorsal protocerebrum. The varicosities contained large dense‐core vesicles (DCVs), and also numerous small clear vesicles, forming divergent output synapses onto unlabeled neurites. The DCVs apparently dock at nonsynaptic sites, suggesting their nonsynaptic release. In addition, a 3D reconstruction revealed the presence of input synapses onto the PDF‐positive fibers. These were detected less frequently than output sites. These observations suggest that the PDF‐positive clock neurons receive neural inputs directly through synaptic connections in the dorsal protocerebrum, in addition to supplying dual outputs, either synaptic or via paracrine release of the DCV contents, to unidentified target neurons. J. Comp. Neurol. 518:292–304, 2010.

Synaptic connections between pigment‐dispersing factor‐immunoreactive neurons and neurons in the pars lateralis of the blow fly Protophormia terraenovae

In females of the blow fly Protophormia terraenovae, neurons with cell bodies in the pars lateralis (PL) projecting to the retrocerebral complex (designated as PL neurons) are necessary for the induction of reproductive diapause under short‐day and low‐temperature conditions. In the present study, neural connections between PL neurons and pigment‐dispersing factor (PDF)‐immunoreactive neurons were examined via immunolight microscopy and immunoelectron microscopy combined with backfills through the cardiac‐recurrent nerve. Immunolight microscopy showed that fibers of PL neurons overlapped with PDF‐immunoreactive fibers in the dorsolateral region of the superior protocerebral neuropil. Immunoelectron microscopy showed that PDF‐immunoreactive fibers formed output synapses with fibers of PL neurons and unlabeled neurons in a region dorsoanteriorly located with respect to the calyx of the mushroom body. The distribution of synaptic connections between PDF‐immunoreactive fibers and the fibers of PL neurons was sparse. According to the projection patterns, PDF‐immunoreactive fibers with synaptic connections with PL neurons appeared to originate from PDF‐immunoreactive neurons with cell bodies at the base of the medulla of the optic lobe (medulla PDF neurons), which are putative circadian clock neurons in P. terraenovae. PDF immunoreactivity was restrictively detected in dense‐core vesicles but not in clear synaptic vesicles. The present results suggest that medulla PDF neurons convey time or photoperiodic information to PL neurons for diapause induction through direct synaptic connections. J. Comp. Neurol. 491:390–399, 2005.

Proctolinergic innervation of the accessory gland in male crickets (Gryllus bimaculatus): Detection of proctolin and some pharmacological properties of myogenically and neurogenically evoked contractions

Abstract Spontaneous contractions were observed in the cricket male accessory gland which is innervated by dorsal unpaired median neurones (DUMR7 neurones) and paired neurones (LC neurones) arising from the terminal abdominal ganglion. These contractions were myogenic: identical mechanical activity was observable in a transplanted accessory gland which lacked innervation. Selective antidromic electrical stimulation of the DUMR7 neurones evoked a contraction of the accessory gland, which was graded and dependent upon both frequency and duration of stimulation. Glutamate at 10−4 M, and proctolin, at low concentrations of 10−9 M, produced sustained contractions of the accessory gland. These drugs at higher concentrations (glutamate, > 10−4 M; proctolin, > 10−8 M) reduced the amplitude of neurogenically evoked contractions and increased the basal tonus. Octopamine, at 10−7 M, increased the frequency of myogenically evoked contractions. Application of serotonin, at 10−5 M, reduced the amplitude of neurally evoked contractions and also resulted in a lowering of the basal tonus. By use of high-performance liquid chromatoraphy and bioassay we have found that proctolin was present in the accessory gland, and it was released from the accessory gland in response to the application of high K+ saline. No proctolin was detected in the transplanted accessory gland.

Visual and olfactory input segregation in the mushroom body calyces in a basal neopteran, the American cockroach.

The cockroach Periplaneta americana is an evolutionary basal neopteran insect, equipped with one of the largest and most elaborate mushroom bodies among insects. Using intracellular recording and staining in the protocerebrum, we discovered two new types of neurons that receive direct input from the optic lobe in addition to the neuron previously reported. These neurons have dendritic processes in the optic lobe, projection sites in the optic tracts, and send axonal terminals almost exclusively to the innermost layer of the MB calyces (input site of MB). Their responses were excitatory to visual but inhibitory to olfactory stimuli, and weak excitation occurred in response to mechanosensory stimuli to cerci. In contrast, interneurons with dendrites mainly in the antennal lobe projection sites send axon terminals to the middle to outer layers of the calyces. These were excited by various olfactory stimuli and mechanosensory stimuli to the antenna. These results suggest that there is general modality-specific terminal segregation in the MB calyces and that this is an early event in insect evolution. Possible postsynaptic and presynaptic elements of these neurons are discussed.

Neural- and endocrine control of flight muscle degeneration in the adult cricket, Gryllus bimaculatus

Neural- and endocrine mechanisms controlling degeneration of a dorsal longitudinal flight muscle, M112a, have been studied in adult Gryllus bimaculatus (Orthoptera: Gryllidae). Decapitation completely prevented muscle degeneration. Implantation of a pair of corpora allata or injection of juvenile hormone III into decapitated crickets caused muscle degeneration. Denervation of M112a resulted in reduction of muscle mass compared with that in sham-operated crickets. Denervation of M112a in decapitated crickets, however, did not affect muscle mass. Birefringence and ultrastructure of M112a showed an obvious regional difference in the onset of degeneration. Fibrillar structures of M112a always disappeared from the ventral to dorsal part. Distribution of axon terminals of motor neurons and mechanical responses to the motor nerve stimuli showed that M112a is composed of five motor units with similar twitch properties. When M112a was fully denervated, regional differences in degeneration disappeared. Partial denervation resulted in denervated muscle fibers losing birefringence earlier than innervated fibers. These results suggest that juvenile hormone causes breakdown of flight muscles, and neural factors control degeneration of flight muscles to some extent under the presence of the juvenile hormone.

Blocking synaptic transmission with tetanus toxin light chain reveals modes of neurotransmission in the PDF-positive circadian clock neurons of Drosophila melanogaster.

Circadian locomotor rhythms of Drosophila melanogaster are controlled by a neuronal circuit composed of approximately 150 clock neurons that are roughly classified into seven groups. In the circuit, a group of neurons expressing pigment-dispersing factor (PDF) play an important role in organizing the pacemaking system. Recent studies imply that unknown chemical neurotransmitter(s) (UNT) other than PDF is also expressed in the PDF-positive neurons. To explore its role in the circadian pacemaker, we examined the circadian locomotor rhythms of pdf-Gal4/UAS-TNT transgenic flies in which chemical synaptic transmission in PDF-positive neurons was blocked by expressed tetanus toxin light chain (TNT). In constant darkness (DD), the flies showed a free-running rhythm, which was similar to that of wild-type flies but significantly different from pdf null mutants. Under constant light conditions (LL), however, they often showed complex rhythms with a short period and a long period component. The UNT is thus likely involved in the synaptic transmission in the clock network and its release caused by LL leads to arrhythmicity. Immunocytochemistry revealed that LL induced phase separation in TIMELESS (TIM) cycling among some of the PDF-positive and PDF-negative clock neurons in the transgenic flies. These results suggest that both PDF and UNT play important roles in the Drosophila circadian clock, and activation of PDF pathway alone by LL leads to the complex locomotor rhythm through desynchronized oscillation among some of the clock neurons.

Development of Invertebrate Brain Platform: Management of Research Resources for Invertebrate Neuroscience and Neuroethology

Various kinds of analysis and mathematical models based on neuroscience are developing in the neural network study. In the research, experimental data and knowledge so far obtained are essential resources to deepen their consideration of neuronal systems and functions. In order to utilize the accumulation of expertise and research effectively, it is important to integrate various resources, such as bibliography and experimental data. The managing and sharing of research resources are absolutely imperative for future development in both experimental and analytical studies. Various scientific fields need a new method to obtain precise information, because the amount of experimental data and publications has increased rapidly due to innovations in measurement, computers and network technologies. Under this situation, an effective resource-managing based on CMS (Content Management System) is introduced here for the laboratory use. We are providing functional modules to manage research resources for neuroinfomatics. As a practical use of these modules, a database system for managing image data of invertebrate neurons measured by Confocal Laser Scanning Microscope (CLSM) is developed. Furthermore, these modules are implemented for management of contents in the Invertebrate Brain Platform.

Localization of RFamide-like Immunoreactivity in the Visceral Organs and Peripheral Neurosecretory Cells Related to the Terminal Abdominal Ganglion in the Cricket, Gryllus bimaculatus

Abstract The distribution of RFamide-like immunoreactivity was examined in the terminal abdominal ganglion (TAG)-related peripheral nervous system, and in the visceral tissues of the female cricket, Gryllus bimaculatus. In the TAG, most of the RFamide-like immunoreactive (RFaLI) neurons were bilaterally paired, and were found in the neuromeres of the 7th to the 10/11th segment. The remaining neurons were found in the midline region. These included anterior unpaired neurons, a cluster of neurons in the posterior end, and ventral paired neurons in the 9th segment. Immunocytochemistry combined with Lucifer Yellow back-filling indicated that the immunoreactive paired medial neurons innervated the rectum. In the visceral tissues innervated by the TAG, RFaLI fibers were seen in the oviducts and rectum, while the spermathecal duct and spermatheca did not exhibit any immunoreactivity. In the peripheral nervous system, RFaLI peripheral neurosecretory cells (PNCs) were found on the fifth and seventh segmental nerve roots of the TAG in both sexes. The perikaryon of the PNCs contained several immunoreactive large electron-dense granular vesicles. Intracellular dye injection showed that the PNCs probably form neurohaemal-release sites for RFamide-like peptide. The PNCs did not project any processes into the TAG.