A descending inhibitory mechanism of nociception mediated by an evolutionarily conserved neuropeptide system in Drosophila.

Nociception is a neural process that animals have developed to avoid potentially tissue-damaging stimuli. While nociception is triggered in the peripheral nervous system, its modulation by the central nervous system is a critical process in mammals, whose dysfunction has been extensively implicated in chronic pain pathogenesis. The peripheral mechanisms of nociception are largely conserved across the animal kingdom. However, it is unclear whether the brain-mediated modulation is also conserved in non-mammalian species. Here, we show that Drosophila has a descending inhibitory mechanism of nociception from the brain, mediated by the neuropeptide Drosulfakinin (DSK), a homolog of cholecystokinin (CCK) that plays an important role in the descending control of nociception in mammals. We found that mutants lacking dsk or its receptors are hypersensitive to noxious heat. Through a combination of genetic, behavioral, histological, and Ca2+ imaging analyses, we subsequently revealed neurons involved in DSK-mediated nociceptive regulation at a single-cell resolution and identified a DSKergic descending neuronal pathway that inhibits nociception. This study provides the first evidence for a descending modulatory mechanism of nociception from the brain in a non-mammalian species that is mediated by the evolutionarily conserved CCK system, raising the possibility that the descending inhibition is an ancient mechanism to regulate nociception.

Avoiding harm is fundamental for the survival of animals. Nerve cells called nociceptors can detect potential damage, such as extreme temperatures, sharp objects and certain chemicals. In humans, this detection ­ known as nociception ­ leads to signals travelling from nociceptors through the spinal cord to the brain, which perceives them as pain. Mammals such as humans and rodents can inhibit nociception by sending signals from the brain to the spinal cord to dampen pain. This top-down dampening process is believed to play a crucial role in regulating pain in mammals, and it has been implicated in the development of chronic pain. It was not known whether non-mammalian animals shared this inhibitory pathway. However, previous work had shown that fruit fly produce a molecule called Drosulfakinin, which is similar to the chemical that mammals use in the top-down signalling pathway which controls pain. To determine the role of Drosulfakinin in controlling fly nociception, Oikawa et al. manipulated its activity ­ and the activity of related genes ­ in specific neurons in the fruit fly nervous system. Without Drosulfakinin, fly larvae were more sensitive to heat exposure, suggesting that this molecule is required to inhibit nociception. Further experiments showed that Drosulfakinin is present only in the brain of fly larvae and activation of its signaling lowers the activity of neurons that transmit nociceptive signals in the insect equivalent of the spinal cord. This confirms that insect brains can dampen nociception via a top-down pathway, using a similar molecule to mammals. The findings provide an important foundation for pain studies using non-mammalian animals. The ability to manipulate nociception using genetic techniques in flies offers a powerful tool to understand the top-down process of controlling pain. This result also raises the possibility that this shared top-down inhibition mechanism may have developed over 550 million years ago, which could lead to further research into how nociception and pain regulation systems evolved.

Transplantation of dental pulp-derived cell sheets cultured on human amniotic membrane induced to differentiate into bone.

OBJECTIVE: The usefulness of the amniotic membrane as a cell culture substrate has led to its use in the development of dental pulp-derived cell sheets. We induced osteoblastic differentiation of dental pulp-derived cell sheets and conducted histological and immunological examinations in addition to imaging assessments for regeneration of bone defects. METHODS: Dental pulp cells were obtained by primary culture of the dental pulp tissue harvested from extracted wisdom teeth. These cells were maintained for three to four passages. Subsequently, the dental pulp cells were seeded onto an amniotic membrane to produce dental pulp-derived cell sheets. Following the induction of osteoblastic differentiation, the sheets were grafted into the subcutaneous tissue of the lower back and maxillary bone defect of a nude mouse. Histological and immunological examinations of both grafts were performed. RESULTS: Dental pulp-derived cell sheets cultured on an osteoblast differentiation-inducing medium demonstrated resemblance to dental pulp tissue and produced calcified tissue. Mineralization was maintained following grafting of the sheets. Regeneration of the maxillary bone defect was observed. CONCLUSION: Induction of osteoblastic differentiation of the dental pulp-derived cell sheets may be indicated for the regeneration of periodontal tissue.

BMP signaling downstream of the Highwire E3 ligase sensitizes nociceptors.

A comprehensive understanding of the molecular machinery important for nociception is essential to improving the treatment of pain. Here, we show that the BMP signaling pathway regulates nociception downstream of the E3 ubiquitin ligase highwire (hiw). hiw loss of function in nociceptors caused antagonistic and pleiotropic phenotypes with simultaneous insensitivity to noxious heat but sensitized responses to optogenetic activation of nociceptors. Thus, hiw functions to both positively and negatively regulate nociceptors. We find that a sensory reception-independent sensitization pathway was associated with BMP signaling. BMP signaling in nociceptors was up-regulated in hiw mutants, and nociceptor-specific expression of hiw rescued all nociception phenotypes including the increased BMP signaling. Blocking the transcriptional output of the BMP pathway with dominant negative Mad suppressed nociceptive hypersensitivity that was induced by interfering with hiw. The up-regulated BMP signaling phenotype in hiw genetic mutants could not be suppressed by mutation in wallenda suggesting that hiw regulates BMP in nociceptors via a wallenda independent pathway. In a newly established Ca2+ imaging preparation, we observed that up-regulated BMP signaling caused a significantly enhanced Ca2+ signal in the axon terminals of nociceptors that were stimulated by noxious heat. This response likely accounts for the nociceptive hypersensitivity induced by elevated BMP signaling in nociceptors. Finally, we showed that 24-hour activation of BMP signaling in nociceptors was sufficient to sensitize nociceptive responses to optogenetically-triggered nociceptor activation without altering nociceptor morphology. Overall, this study demonstrates the previously unrevealed roles of the Hiw-BMP pathway in the regulation of nociception and provides the first direct evidence that up-regulated BMP signaling physiologically sensitizes responses of nociceptors and nociception behaviors.

Nociceptive interneurons control modular motor pathways to promote escape behavior in Drosophila.

Rapid and efficient escape behaviors in response to noxious sensory stimuli are essential for protection and survival. Yet, how noxious stimuli are transformed to coordinated escape behaviors remains poorly understood. In Drosophila larvae, noxious stimuli trigger sequential body bending and corkscrew-like rolling behavior. We identified a population of interneurons in the nerve cord of Drosophila, termed Down-and-Back (DnB) neurons, that are activated by noxious heat, promote nociceptive behavior, and are required for robust escape responses to noxious stimuli. Electron microscopic circuit reconstruction shows that DnBs are targets of nociceptive and mechanosensory neurons, are directly presynaptic to pre-motor circuits, and link indirectly to Goro rolling command-like neurons. DnB activation promotes activity in Goro neurons, and coincident inactivation of Goro neurons prevents the rolling sequence but leaves intact body bending motor responses. Thus, activity from nociceptors to DnB interneurons coordinates modular elements of nociceptive escape behavior.

Genetic interaction of DISC1 and Neurexin in the development of fruit fly glutamatergic synapses.

Originally identified at the breakpoint of a (1;11)(q42.1; q14.3) chromosomal translocation in a Scottish family with a wide range of mental disorders, the DISC1 gene has been a focus of intensive investigations as an entry point to study the molecular mechanisms of diverse mental dysfunctions. Perturbations of the DISC1 functions lead to behavioral changes in animal models, which are relevant to psychiatric conditions in patients. In this work, we have expressed the human DISC1 gene in the fruit fly (Drosophila melanogaster) and performed a genetic screening for the mutations of psychiatric risk genes that cause modifications of DISC1 synaptic phenotypes at the neuromuscular junction. We found that DISC1 interacts with dnrx1, the Drosophila homolog of the human Neurexin (NRXN1) gene, in the development of glutamatergic synapses. While overexpression of DISC1 suppressed the total bouton area on the target muscles and stimulated active zone density in wild-type background, a partial reduction of the dnrx1 activity negated the DISC1-mediated synaptic alterations. Likewise, overexpression of DISC1 stimulated the expression of a glutamate receptor component, DGLURIIA, in wild-type background but not in the dnrx1 heterozygous background. In addition, DISC1 caused mislocalization of Discs large, the Drosophila PSD-95 homolog, in the dnrx1 heterozygous background. Analyses with a series of domain deletions have revealed the importance of axonal localization of the DISC1 protein for efficient suppression of DNRX1 in synaptic boutons. These results thus suggest an intriguing converging mechanism controlled by the interaction of DISC1 and Neurexin in the developing glutamatergic synapses.

Artificial Induction of Associative Olfactory Memory by Optogenetic and Thermogenetic Activation of Olfactory Sensory Neurons and Octopaminergic Neurons in Drosophila Larvae.

The larval brain of Drosophila melanogaster provides an excellent system for the study of the neurocircuitry mechanism of memory. Recent development of neurogenetic techniques in fruit flies enables manipulations of neuronal activities in freely behaving animals. This protocol describes detailed steps for artificial induction of olfactory associative memory in Drosophila larvae. In this protocol, the natural reward signal is substituted by thermogenetic activation of octopaminergic neurons in the brain. In parallel, the odor signal is substituted by optogenetic activation of a specific class of olfactory receptor neurons. Association of reward and odor stimuli is achieved with the concomitant application of blue light and heat that leads to activation of both sets of neurons in living transgenic larvae. Given its operational simplicity and robustness, this method could be utilized to further our knowledge on the neurocircuitry mechanism of memory in the fly brain.

Nociceptor-Enriched Genes Required for Normal Thermal Nociception.

Here, we describe a targeted reverse genetic screen for thermal nociception genes in Drosophila larvae. Using laser capture microdissection and microarray analyses of nociceptive and non-nociceptive neurons, we identified 275 nociceptor-enriched genes. We then tested the function of the enriched genes with nociceptor-specific RNAi and thermal nociception assays. Tissue-specific RNAi targeted against 14 genes caused insensitive thermal nociception while targeting of 22 genes caused hypersensitive thermal nociception. Previously uncategorized genes were named for heat resistance (i.e., boilerman, fire dancer, oven mitt, trivet, thawb, and bunker gear) or heat sensitivity (firelighter, black match, eucalyptus, primacord, jet fuel, detonator, gasoline, smoke alarm, and jetboil). Insensitive nociception phenotypes were often associated with severely reduced branching of nociceptor neurites and hyperbranched dendrites were seen in two of the hypersensitive cases. Many genes that we identified are conserved in mammals.

Biomechanical force induces the growth factor production in human periodontal ligament-derived cells.

Although many reports have been published on the functional roles of periodontal ligament (PDL) cells, the mechanisms involved in the maintenance and homeostasis of PDL have not been determined. We investigated the effects of biomechanical force on growth factor production, phosphorylation of MAPKs, and intracellular transduction pathways for growth factor production in human periodontal ligament (hPDL) cells using MAPK inhibitors. hPDL cells were exposed to mechanical force (6 MPa) using a hydrostatic pressure apparatus. The levels of growth factor mRNA and protein were examined by real-time RT-PCR and ELISA. The phosphorylation of MAPKs was measured using BD™ CBA Flex Set. In addition, MAPKs inhibitors were used to identify specific signal transduction pathways. Application of biomechanical force (equivalent to occlusal force) increased the synthesis of VEGF-A, FGF-2, and NGF. The application of biomechanical force increased the expression levels of phosphorylated ERK and p38, but not of JNK. Furthermore, the levels of VEGF-A and NGF expression were suppressed by ERK or p38 inhibitor. The growth factors induced by biomechanical force may play a role in the mechanisms of homeostasis of PDL.

Evaluation of a dental pulp-derived cell sheet cultured on amniotic membrane substrate.

OBJECTIVE: Mesenchymal stem cells (MSC) are transplanted for periodontal tissue regeneration, and the periodontal ligament (PDL) is regenerated using a cultured cell sheet. This cultured cell sheet is prepared using PDL-derived cells, growth factors, and amniotic membrane (AM). Dental pulp (DP)-derived cells can be easily obtained from extracted wisdom teeth, proliferate rapidly, and are less susceptible to bacterial infection than PDL-derived cells. Thus, to prepare a novel cell sheet, DP-derived cells were cultured on AM as a culture substrate for immunohistochemical examination. METHODS: Wisdom teeth extracted from three adults were cut along the cement-enamel border. DP tissue was collected, minced, and primarily cultured. After three or four passage cultures, DP-derived cells were cultured on AM, followed by hematoxylin-eosin (H-E) and immunofluorescence staining. RESULTS: DP-derived cells cultured on AM formed a layered structure. Cells positive for vimentin, Ki-67, ZO-1, desmoplakin, CD29, 44, 105 or 146, STRO-1, collagen IV or VII or laminin 5 or α5 chain were localized. CONCLUSIONS: DP-derived cells proliferated on AM, while retaining the properties of DP, which allowed the cultured cell sheet to be prepared. In addition, the cultured cell sheet contained MSC, which suggests its potential application in periodontal tissue regeneration.

Balboa binds to pickpocket in vivo and is required for mechanical nociception in Drosophila larvae.

The Drosophila gene pickpocket (ppk) encodes an ion channel subunit of the degenerin/epithelial sodium channel (DEG/ENaC) family. PPK is specifically expressed in nociceptive, class IV multidendritic (md) neurons and is functionally required for mechanical nociception responses. In this study, in a genome-wide genetic screen for other ion channel subunits required for mechanical nociception, we identify a gene that we name balboa (also known as CG8546, ppk26). Interestingly, the balboa locus encodes a DEG/ENaC ion channel subunit highly similar in amino acid sequence to PPK. Moreover, laser-capture isolation of RNA from larval neurons and microarray analyses reveal that balboa is also highly enriched in nociceptive neurons. The requirement for Balboa and PPK in mechanical nociception behaviors and their specific expression in larval nociceptors led us to hypothesize that these DEG/ENaC subunits form an ion channel complex in vivo. In nociceptive neurons, Balboa::GFP proteins distribute uniformly throughout dendrites but remarkably localize to discrete foci when ectopically expressed in other neuron subtypes (where PPK is not expressed). Indeed, ectopically coexpressing ppk transforms this punctate Balboa::GFP expression pattern to the uniform distribution observed in its native cell type. Furthermore, ppk-RNAi in class IV neurons alters the broad Balboa::GFP pattern to a punctate distribution. Interestingly, this interaction is mutually codependent as balboa-RNAi eliminates Venus::PPK from the sensory dendrites of nociceptors. Finally, using a GFP-reconstitution approach in transgenic larvae, we directly detect in vivo physical interactions among PPK and Balboa subunits. Combined, our results indicate a critical mechanical nociception function for heteromeric PPK and Balboa channels in vivo.

Induction of associative olfactory memory by targeted activation of single olfactory neurons in Drosophila larvae.

It has been postulated that associative memory is formed by at least two sets of external stimuli, CS and US, that are transmitted to the memory centers by distinctive conversing pathways. However, whether associative memory can be induced by the activation of only the olfactory CS and a biogenic amine-mediated US pathways remains to be elucidated. In this study, we substituted the reward signals with dTrpA1-mediated thermogenetic activation of octopaminergic neurons and the odor signals by ChR2-mediated optical activation of a specific class of olfactory neurons. We show that targeted activation of the olfactory receptor and the octopaminergic neurons is indeed sufficient for the formation of associative olfactory memory in the larval brain. We also show that targeted stimulation of only a single type of olfactory receptor neurons is sufficient to induce olfactory memory that is indistinguishable from natural memory induced by the activation of multiple olfactory receptor neurons.

ß-cryptoxanthin regulates bone resorption related-cytokine production in human periodontal ligament cells.

OBJECTIVE: ß-cryptoxanthin (ß-cry) is a type of carotenoid found in certain fruits and vegetables. Although it has been shown that ß-cry inhibits alveolar bone resorption, the molecular mechanisms for this have not yet been clarified. In the present study, we investigated the effects of ß-cry on bone resorption related-cytokine production in human periodontal ligament (hPDL) cells. DESIGN: hPDL cells were stimulated with ß-cry (1×10(-7)mol/l), mechanical stress (1 or 6MPa), and P. gingivalis. The production of interleukin (IL)-1ß, IL-6, IL-8, tumour necrosis factor (TNF)-α, osteoprotegerin (OPG), and receptor activator of nuclear factor kappa-B ligand (RANKL) were analyzed by RT-PCR and ELISA. RESULTS: The production of IL-1ß, IL-6, IL-8, and TNF-α was not induced in hPDL cells after stimulation with ß-cry, although these cytokines were produced after stimulation with P. gingivalis. On the other hand, IL-6 and IL-8 were produced after exposure to 6MPa of mechanical stress. The production of IL-6 and IL-8 was significantly decreased by the addition of ß-cry. Furthermore, ß-cry up-regulated the production of OPG, but not RANKL. CONCLUSION: ß-cry inhibited the production of IL-6 and IL-8 induced by mechanical stress and periodontopathogenic bacteria in hPDL cells. Moreover, ß-cry up-regulated OPG production. These results suggest that ß-cry may prevent bone resorption in periodontitis.

Optogenetic manipulation of neural circuits and behavior in Drosophila larvae.

Optogenetics is a powerful tool that enables the spatiotemporal control of neuronal activity and circuits in behaving animals. Here, we describe our protocol for optical activation of neurons in Drosophila larvae. As an example, we discuss the use of optogenetics to activate larval nociceptors and nociception behaviors in the third-larval instar. We have previously shown that, using spatially defined GAL4 drivers and potent UAS (upstream activation sequence)-channelrhodopsin-2∷YFP transgenic strains developed in our laboratory, it is possible to manipulate neuronal populations in response to illumination by blue light and to test whether the activation of defined neural circuits is sufficient to shape behaviors of interest. Although we have only used the protocol described here in larval stages, the procedure can be adapted to study neurons in adult flies--with the caveat that blue light may not sufficiently penetrate the adult cuticle to stimulate neurons deep in the brain. This procedure takes 1 week to culture optogenetic flies and ~1 h per group for the behavioral assays.

Preventive effects of a kampo medicine, orento on inflammatory responses in lipopolysaccharide treated human gingival fibroblasts.

In the present study, we investigated the effects of a Kampo medicine Orento (TJ-120) on the production of prostaglandin E(2) (PGE(2)), interleukin (IL)-6 and IL-8 by human gingival fibroblasts (HGFs) treated with lipopolysaccharide from Porphyromonas gingivalis (PgLPS). HGFs proliferation was dose-dependently decreased with Orento at days 3 and 7. However, treatment with PgLPS (10 ng/ml), Orento (up to 1 mg/ml) and their combinations for 24 h did not affect the viability of HGFs. Orento suppressed PgLPS-induced PGE(2) production in a dose-dependent manner but did not alter basal PGE(2) level. In contrast, Orento did not alter PgLPS-induced IL-6 and IL-8 productions. These alterations by Orento were similar to those by a mitogen-activated protein kinase kinase (MAPKK/MEK) inhibitor, PD98059. A Orento showed no effect on cyclooxygenase (COX)-1 and COX-2 activities, and increased cytoplasmic phospholipase A(2) (cPLA(2)) expression and increased PgLPS-induced COX-2 expression. Orento suppressed PgLPS-induced mobility retardation of cPLA(2) band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, that is cPLA(2) phosphorylation and its activation, while Orento alone did not alter cPLA(2) phosphorylation. Orento suppressed PgLPS-induced extracellular signal-regulated kinase (ERK) phosphorylation, which is known to lead to ERK activation and cPLA(2) phosphorylation. These results suggest that Orento decreased PGE(2) production by inhibition of cPLA(2) phosphorylation and its activation via inhibition of ERK phosphorylation, and also that Orento may be useful to improve gingival inflammation in periodontal disease.

Distinctive neuronal networks and biochemical pathways for appetitive and aversive memory in Drosophila larvae.

Associative strength between conditioned stimulus (CS) and unconditioned stimulus (US) is thought to determine learning efficacy in classical conditioning. Elucidation of the neuronal mechanism that underlies the association between CS and US in the brain is thus critical to understand the principle of memory formation. With a simple brain organization, the Drosophila larva provides an attractive model system to investigate learning at the neurocircuitry level. Previously, we described a single-odor paradigm for larval associative learning using sucrose as a reward, and showed that larval appetitive memory lasts longer than 2 h. In this work, we describe behavioral and genetic characterization of larval aversive olfactory memory formed in our paradigm, and compare its stability and neurocircuitry with those of appetitive memory. Despite identical training paradigms, larval olfactory memory formed with quinine or NaCl is short-lived to be lost in 20 min. As with appetitive memory, larval aversive memory produced in this paradigm depends on intact cAMP signaling, but neither mutation of amnesiac nor suppression of CREB activity affects its kinetics. Neurocircuitry analyses suggest that aversive memory is stored before the presynaptic termini of the larval mushroom body neurons as is the case with appetitive memory. However, synaptic output of octopaminergic and dopaminergic neurons, which exhibit distinctive innervation patterns on the larval mushroom body and antennal lobe, is differentially required for the acquisition of appetitive and aversive memory, respectively. These results as a whole suggest that the genetically programmed memory circuitries might provide predisposition in the efficacy of inducing longer-lived memory components in associative learning.

Differential microarray analysis of Drosophila mushroom body transcripts using chemical ablation.

Mushroom bodies (MBs) are the centers for olfactory associative learning and elementary cognitive functions in the Drosophila brain. As a way to systematically elucidate genes preferentially expressed in MBs, we have analyzed genome-wide alterations in transcript profiles associated with MB ablation by hydroxyurea. We selected 100 genes based on microarray data and examined their expression patterns in the brain by in situ hybridization. Seventy genes were found to be expressed in the posterodorsal cortex, which harbors the MB cell bodies. These genes encode proteins of diverse functions, including transcription, signaling, cell adhesion, channels, and transporters. Moreover, we have examined developmental functions of 40 of the microarray-identified genes by transgenic RNA interference; 8 genes were found to cause mild-to-strong MB defects when suppressed with a MB-Gal4 driver. These results provide important information not only on the repertoire of genes that control MB development but also on the repertoire of neural factors that may have important physiological functions in MB plasticity.

Induction of cAMP response element-binding protein-dependent medium-term memory by appetitive gustatory reinforcement in Drosophila larvae.

The fruit fly Drosophila melanogaster has been successfully used as a model animal for the study of the genetic and molecular mechanisms of learning and memory. Although most of the Drosophila learning studies have used the adult fly, the relative complexity of its neural network hinders cellular and molecular studies at high resolution. In contrast, the Drosophila larva has a simple brain with uniquely identifiable neural networks, providing an opportunity of an attractive alternative system for elucidation of underlying mechanisms involved in learning and memory. In this paper, we describe a novel paradigm of larval associative learning with a single odor and a positive gustatory reinforcer, sucrose. Mutant analyses have suggested importance of cAMP signaling and potassium channel activities in larval learning as has been demonstrated with the adult fly. Intriguingly, larval memory produced by the appetitive conditioning lasts medium term and depends on both amnesiac and cAMP response element-binding protein (CREB). A significant part of memory was disrupted at very early phase by CREB blockade without affecting immediate learning performance. Moreover, we also show that synaptic output of larval mushroom body neurons is required for retrieval but not for acquisition and retention of the larval memory, including the CREB-dependent component.