Error Function Optimization to Compare Neural Activity and Train Blended Rhythmic Networks.

We present a novel set of quantitative measures for "likeness" (error function) designed to alleviate the time-consuming and subjective nature of manually comparing biological recordings from electrophysiological experiments with the outcomes of their mathematical models. Our innovative "blended" system approach offers an objective, high-throughput, and computationally efficient method for comparing biological and mathematical models. This approach involves using voltage recordings of biological neurons to drive and train mathematical models, facilitating the derivation of the error function for further parameter optimization. Our calibration process incorporates measurements such as action potential (AP) frequency, voltage moving average, voltage envelopes, and the probability of post-synaptic channels. To assess the effectiveness of our method, we utilized the sea slug Melibe leonina swim central pattern generator (CPG) as our model circuit and conducted electrophysiological experiments with TTX to isolate CPG interneurons. During the comparison of biological recordings and mathematically simulated neurons, we performed a grid search of inhibitory and excitatory synapse conductance. Our findings indicate that a weighted sum of simple functions is essential for comprehensively capturing a neuron's rhythmic activity. Overall, our study suggests that our blended system approach holds promise for enabling objective and high-throughput comparisons between biological and mathematical models, offering significant potential for advancing research in neural circuitry and related fields.

Cold-Temperature Coding with Bursting and Spiking Based on TRP Channel Dynamics in Drosophila Larva Sensory Neurons.

Temperature sensation involves thermosensitive TRP (thermoTRP) and non-TRP channels. Drosophila larval Class III (CIII) neurons serve as the primary cold nociceptors and express a suite of thermoTRP channels implicated in noxious cold sensation. How CIII neurons code temperature remains unclear. We combined computational and electrophysiological methods to address this question. In electrophysiological experiments, we identified two basic cold-evoked patterns of CIII neurons: bursting and spiking. In response to a fast temperature drop to noxious cold, CIII neurons distinctly mark different phases of the stimulus. Bursts frequently occurred along with the fast temperature drop, forming a peak in the spiking rate and likely coding the high rate of the temperature change. Single spikes dominated at a steady temperature and exhibited frequency adaptation following the peak. When temperature decreased slowly to the same value, mainly spiking activity was observed, with bursts occurring sporadically throughout the stimulation. The spike and the burst frequencies positively correlated with the rate of the temperature drop. Using a computational model, we explain the distinction in the occurrence of the two CIII cold-evoked patterns bursting and spiking using the dynamics of a thermoTRP current. A two-parameter activity map (Temperature, constant TRP current conductance) marks parameters that support silent, spiking, and bursting regimes. Projecting on the map the instantaneous TRP conductance, governed by activation and inactivation processes, reflects temperature coding responses as a path across silent, spiking, or bursting domains on the map. The map sheds light on how various parameter sets for TRP kinetics represent various types of cold-evoked responses. Together, our results indicate that bursting detects the high rate of temperature change, whereas tonic spiking could reflect both the rate of change and magnitude of steady cold temperature.

Regulatory Aspects of Cell and Gene Therapy Products: The Japanese Perspective.

Regulations for regenerative medicine for human use, such as cell and gene therapy (CGT), have evolved in accordance with advancements in clinical experience, scientific knowledge, and social acceptance of these technologies. In November 2014, two acts, "The Act on the Safety of Regenerative Medicine" (ASRM) and the "Pharmaceuticals, Medical Devices, and Other Therapeutic Products Act" (PMD Act), came into effect in Japan. The responsibilities of medical institutions in ensuring the safety and transparency of such medical technologies are described under ASRM. The PMD Act provides the option of a new scheme for obtaining conditional and time-limited approval for CGT products. Overall, research and development on CGT products, especially gene therapy products, is progressing. New legislative frameworks have been designed to promote the timely development of new technologies and safe and effective CGT products for Japanese patients.

Regulatory Issues: PMDA - Review of Sakigake Designation Products: Oncolytic Virus Therapy with Delytact Injection (Teserpaturev) for Malignant Glioma.

In June 2021, the Ministry of Health, Labor and Welfare approved Delytact Injection as a regenerative medical product for oncolytic virus therapy. The active substance of Delytact Injection is teserpaturev, a genetically engineered herpes simplex virus type 1 (strain F) in which the α47 gene and both copies of the γ34.5 gene have been deleted and the infected cell protein 6 (ICP6) gene has been inactivated by the insertion of the lacZ gene from Escherichia coli. Delytact Injection, when intratumorally administered to patients with malignant glioma, is expected to exert the following effects: (1) the mutant virus selectively replicates in tumor cells and destroys the infected cells through the replication process, exerting a cytocidal effect, and (2) the administration leads to induction of tumor-responsive T cells, which activates antitumor immunity and thus prolongs the survival of patients with malignant glioma. A Japanese phase II study (Study GD01) was conducted in patients with glioblastoma who had residual or recurrent tumors after radiotherapy with concomitant temozolomide. In Study GD01, however, stable disease continued for an extended period in some patients with glioblastoma. Hence, Delytact Injection is expected to be effective to a certain level. In line with this, Delytact Injection has been approved as an option for the treatment of malignant glioma, with one of the 3 approval conditions including conducting a use-results comparison survey and resubmission of the marketing authorization application within the granted time period of 7 years, under the conditional and time-limited approval scheme described in Article 23-26 of Act on Securing Quality, Efficacy and Safety of Products Including Pharmaceuticals and Medical Devices.

Chloride-dependent mechanisms of multimodal sensory discrimination and nociceptive sensitization in Drosophila.

Individual sensory neurons can be tuned to many stimuli, each driving unique, stimulus-relevant behaviors, and the ability of multimodal nociceptor neurons to discriminate between potentially harmful and innocuous stimuli is broadly important for organismal survival. Moreover, disruptions in the capacity to differentiate between noxious and innocuous stimuli can result in neuropathic pain. Drosophila larval class III (CIII) neurons are peripheral noxious cold nociceptors and innocuous touch mechanosensors; high levels of activation drive cold-evoked contraction (CT) behavior, while low levels of activation result in a suite of touch-associated behaviors. However, it is unknown what molecular factors underlie CIII multimodality. Here, we show that the TMEM16/anoctamins subdued and white walker (wwk; CG15270) are required for cold-evoked CT, but not for touch-associated behavior, indicating a conserved role for anoctamins in nociception. We also evidence that CIII neurons make use of atypical depolarizing chloride currents to encode cold, and that overexpression of ncc69-a fly homologue of NKCC1-results in phenotypes consistent with neuropathic sensitization, including behavioral sensitization and neuronal hyperexcitability, making Drosophila CIII neurons a candidate system for future studies of the basic mechanisms underlying neuropathic pain.

Modality specific roles for metabotropic GABAergic signaling and calcium induced calcium release mechanisms in regulating cold nociception.

Calcium (Ca2+) plays a pivotal role in modulating neuronal-mediated responses to modality-specific sensory stimuli. Recent studies in Drosophila reveal class III (CIII) multidendritic (md) sensory neurons function as multimodal sensors regulating distinct behavioral responses to innocuous mechanical and nociceptive thermal stimuli. Functional analyses revealed CIII-mediated multimodal behavioral output is dependent upon activation levels with stimulus-evoked Ca2+ displaying relatively low vs. high intracellular levels in response to gentle touch vs. noxious cold, respectively. However, the mechanistic bases underlying modality-specific differential Ca2+ responses in CIII neurons remain incompletely understood. We hypothesized that noxious cold-evoked high intracellular Ca2+ responses in CIII neurons may rely upon Ca2+ induced Ca2+ release (CICR) mechanisms involving transient receptor potential (TRP) channels and/or metabotropic G protein coupled receptor (GPCR) activation to promote cold nociceptive behaviors. Mutant and/or CIII-specific knockdown of GPCR and CICR signaling molecules [GABA B -R2, Gαq, phospholipase C, ryanodine receptor (RyR) and Inositol trisphosphate receptor (IP3R)] led to impaired cold-evoked nociceptive behavior. GPCR mediated signaling, through GABA B -R2 and IP3R, is not required in CIII neurons for innocuous touch evoked behaviors. However, CICR via RyR is required for innocuous touch-evoked behaviors. Disruptions in GABA B -R2, IP3R, and RyR in CIII neurons leads to significantly lower levels of cold-evoked Ca2+ responses indicating GPCR and CICR signaling mechanisms function in regulating Ca2+ release. CIII neurons exhibit bipartite cold-evoked firing patterns, where CIII neurons burst during rapid temperature change and tonically fire during steady state cold temperatures. GABA B -R2 knockdown in CIII neurons resulted in disorganized firing patterns during cold exposure. We further demonstrate that application of GABA or the GABA B specific agonist baclofen potentiates cold-evoked CIII neuron activity. Upon ryanodine application, CIII neurons exhibit increased bursting activity and with CIII-specific RyR knockdown, there is an increase in cold-evoked tonic firing and decrease in bursting. Lastly, our previous studies implicated the TRPP channel Pkd2 in cold nociception, and here, we show that Pkd2 and IP3R genetically interact to specifically regulate cold-evoked behavior, but not innocuous mechanosensation. Collectively, these analyses support novel, modality-specific roles for metabotropic GABAergic signaling and CICR mechanisms in regulating intracellular Ca2+ levels and cold-evoked behavioral output from multimodal CIII neurons.

Transient and Steady-State Properties of Drosophila Sensory Neurons Coding Noxious Cold Temperature.

Coding noxious cold signals, such as the magnitude and rate of temperature change, play essential roles in the survival of organisms. We combined electrophysiological and computational neuroscience methods to investigate the neural dynamics of Drosophila larva cold-sensing Class III (CIII) neurons. In response to a fast temperature change (-2 to -6°C/s) from room temperature to noxious cold, the CIII neurons exhibited a pronounced peak of a spiking rate with subsequent relaxation to a steady-state spiking. The magnitude of the peak was higher for a higher rate of temperature decrease, while slow temperature decrease (-0.1°C/s) evoked no distinct peak of the spiking rate. The rate of the steady-state spiking depended on the magnitude of the final temperature and was higher at lower temperatures. For each neuron, we characterized this dependence by estimating the temperature of the half activation of the spiking rate by curve fitting neuron's spiking rate responses to a Boltzmann function. We found that neurons had a temperature of the half activation distributed over a wide temperature range. We also found that CIII neurons responded to decrease rather than increase in temperature. There was a significant difference in spiking activity between fast and slow returns from noxious cold to room temperature: The CIII neurons usually stopped activity abruptly in the case of the fast return and continued spiking for some time in the case of the slow return. We developed a biophysical model of CIII neurons using a generalized description of transient receptor potential (TRP) current kinetics with temperature-dependent activation and Ca2+-dependent inactivation. This model recapitulated the key features of the spiking rate responses found in experiments and suggested mechanisms explaining the transient and steady-state activity of the CIII neurons at different cold temperatures and rates of their decrease and increase. We conclude that CIII neurons encode at least three types of cold sensory information: the rate of temperature decrease by a peak of the firing rate, the magnitude of cold temperature by the rate of steady spiking activity, and direction of temperature change by spiking activity augmentation or suppression corresponding to temperature decrease and increase, respectively.

Protocols for measuring cold-evoked neural activity and cold tolerance in Drosophila larvae following fictive cold acclimation.

Here, we outline protocols to study cold acclimation in Drosophila from a neurobiological perspective, starting with fictive cold acclimation using a custom-built optogenetics-housing apparatus we call the OptoBox. We also provide detailed steps for single-unit electrophysiological recordings from larval cold nociceptors and a high-throughput cold-tolerance assay. These protocols expand the toolkit for the study of insect cold acclimation and nociception. For complete details on the use and execution of this protocol, please refer to Himmel et al. (2021).

The Consistency of Gastropod Identified Neurons Distinguishes Intra-Individual Plasticity From Inter-Individual Variability in Neural Circuits.

Gastropod mollusks are known for their large, individually identifiable neurons, which are amenable to long-term intracellular recordings that can be repeated from animal to animal. The constancy of individual neurons can help distinguish state-dependent or temporal variation within an individual from actual variability between individual animals. Investigations into the circuitry underlying rhythmic swimming movements of the gastropod species, Tritonia exsulans and Pleurobranchaea californica have uncovered intra- and inter-individual variability in synaptic connectivity and serotonergic neuromodulation. Tritonia has a reliably evoked escape swim behavior that is produced by a central pattern generator (CPG) composed of a small number of identifiable neurons. There is apparent individual variability in some of the connections between neurons that is inconsequential for the production of the swim behavior under normal conditions, but determines whether that individual can swim following a neural lesion. Serotonergic neuromodulation of synaptic strength intrinsic to the CPG creates neural circuit plasticity within an individual and contributes to reorganization of the network during recovery from injury and during learning. In Pleurobranchaea, variability over time in the modulatory actions of serotonin and in expression of serotonin receptor genes in an identified neuron directly reflects variation in swimming behavior. Tracking behavior and electrophysiology over hours to days was necessary to identify the functional consequences of these intra-individual, time-dependent variations. This work demonstrates the importance of unambiguous neuron identification, properly assessing the animal and network states, and tracking behavior and physiology over time to distinguish plasticity within the same animal at different times from variability across individual animals.

Bursting emerges from the complementary roles of neurons in a four-cell network.

Reciprocally inhibitory modules that form half-center oscillators require mechanisms for escaping or being released from inhibition. The central pattern generator underlying swimming by the nudibranch mollusc, Dendronotus iris, is composed of only four neurons that are organized into two competing modules of a half-center oscillator. In this system, bursting activity in left-right alternation is an emergent property of the network as a whole; none of the neurons produces bursts on its own. We found that the unique synaptic actions and membrane properties of the two neurons in each module (Si2 and the contralateral Si3) play complementary roles in generating stable bursting in this network oscillator. Although Si2 and Si3 each inhibits its contralateral counterpart, Si2 plays a dominant role in evoking fast and strong inhibition of the other module, the termination of which initiates postinhibitory rebound in the Si3 of that module by activating a hyperpolarization-activated inward current. Within each module, the synaptic actions and membrane properties of the two neurons complement each other: Si3 excites Si2, which then feeds back slow inhibition to Si3, terminating the burst. Using dynamic clamp, we showed that the magnitude of the slow inhibition sets the period of the oscillator. Thus, the synaptic actions of Si2 provide the hyperpolarization needed for the other module to rebound stably, whereas the membrane properties of Si3 in each module cause it to rebound first and excite Si2 to maintain the burst until terminated by the slow inhibition from Si2, which releases the other module to become active.NEW & NOTEWORTHY Half-center oscillators composed of reciprocally inhibitory neurons have been posited for over a century to underlie the production of rhythmic movements. The Dendronotus swim central pattern generator may be the simplest such circuit with only two pairs of bilaterally represented neurons. This study completes the description of the mechanism by which this network oscillator functions, showing how stable rhythmic activity arises from the complementary membrane and synaptic properties of the two neurons in the competing modules.

Alteration in information flow through a pair of feeding command neurons underlies a form of Pavlovian conditioning in the Drosophila brain.

Pavlovian conditioning1 is a broadly used learning paradigm where defined stimuli are associated to induce behavioral switching. To define a causal relationship between activity change in a single neuron and behavioral switching, we took advantage of a "command neuron" that connects cellular function to behavior.2 To examine the cellular and molecular basis of Pavlovian conditioning, we previously identified a pair of feeding command neurons termed "feeding neurons" in the adult Drosophila brain3 using genetic screening4 and opto- and thermo-genetic techniques.5-7 The feeding neuron is activated by sweet signals like sucrose and induces the full complement of feeding behaviors, such as proboscis extension and food pumping. Ablation or inactivation of the pair of feeding neurons abolishes feeding behavior, suggesting that this single pair of neurons is indispensable for natural feeding behaviors.2,3 Here, we describe a novel conditioning protocol to associate a signal-mediating rod removal from legs (conditioned stimulus [CS]) to feeding behavior induced by sucrose stimulation (unconditioned stimulus [US]). Calcium imaging of the feeding neuron demonstrated it acquires responsiveness to CS during conditioning, with inactivation of the feeding neuron during conditioning suppressing plasticity. These results suggest conditioning alters signals flowing from the CS into the feeding circuit, with the feeding neuron functioning as a key integrative hub for Hebbian plasticity.

Identification of a neural basis for cold acclimation in Drosophila larvae.

Low temperatures can be fatal to insects, but many species have evolved the ability to cold acclimate, thereby increasing their cold tolerance. It has been previously shown that Drosophila melanogaster larvae perform cold-evoked behaviors under the control of noxious cold-sensing neurons (nociceptors), but it is unknown how the nervous system might participate in cold tolerance. Herein, we describe cold-nociceptive behavior among 11 drosophilid species; we find that the predominant cold-evoked larval response is a head-to-tail contraction behavior, which is likely inherited from a common ancestor, but is unlikely to be protective. We therefore tested the hypothesis that cold nociception functions to protect larvae by triggering cold acclimation. We found that Drosophila melanogaster Class III nociceptors are sensitized by and critical to cold acclimation and that cold acclimation can be optogenetically evoked, sans cold. Collectively, these findings demonstrate that cold nociception constitutes a peripheral neural basis for Drosophila larval cold acclimation.

Synaptotagmin 7 switches short-term synaptic plasticity from depression to facilitation by suppressing synaptic transmission.

Short-term synaptic plasticity is a fast and robust modification in neuronal presynaptic output that can enhance release strength to drive facilitation or diminish it to promote depression. The mechanisms that determine whether neurons display short-term facilitation or depression are still unclear. Here we show that the Ca2+-binding protein Synaptotagmin 7 (Syt7) determines the sign of short-term synaptic plasticity by controlling the initial probability of synaptic vesicle (SV) fusion. Electrophysiological analysis of Syt7 null mutants at Drosophila embryonic neuromuscular junctions demonstrate loss of the protein converts the normally observed synaptic facilitation response during repetitive stimulation into synaptic depression. In contrast, overexpression of Syt7 dramatically enhanced the magnitude of short-term facilitation. These changes in short-term plasticity were mirrored by corresponding alterations in the initial evoked response, with SV release probability enhanced in Syt7 mutants and suppressed following Syt7 overexpression. Indeed, Syt7 mutants were able to display facilitation in lower [Ca2+] where release was reduced. These data suggest Syt7 does not act by directly sensing residual Ca2+ and argues for the existence of a distinct Ca2+ sensor beyond Syt7 that mediates facilitation. Instead, Syt7 normally suppresses synaptic transmission to maintain an output range where facilitation is available to the neuron.

Regulatory aspects of quality and safety for live recombinant viral vaccines against infectious diseases in Japan.

Recombinant viral vaccines expressing antigens of pathogenic microbes (e.g., HIV, Ebola virus, and malaria) have been designed to overcome the insufficient immune responses induced by the conventional vaccines. Our knowledge of and clinical experience with the new recombinant viral vaccines are insufficient, and a clear regulatory pathway is needed for the further development and evaluation of recombinant viral vaccines. In 2018, the research group supported by the Ministry of Health, Labour and Welfare, Japan (MHLW) published a concept paper to address the development of recombinant viral vaccines against infectious diseases. Herein we summarize the concept paper-which explains the Japanese regulatory concerns about recombinant viral vaccines-and provide a focus of discussion about the development of recombinant viral vaccines.

Drosophila menthol sensitivity and the Precambrian origins of transient receptor potential-dependent chemosensation.

Transient receptor potential (TRP) cation channels are highly conserved, polymodal sensors which respond to a wide variety of stimuli. Perhaps most notably, TRP channels serve critical functions in nociception and pain. A growing body of evidence suggests that transient receptor potential melastatin (TRPM) and transient receptor potential ankyrin (TRPA) thermal and electrophile sensitivities predate the protostome-deuterostome split (greater than 550 Ma). However, TRPM and TRPA channels are also thought to detect modified terpenes (e.g. menthol). Although terpenoids like menthol are thought to be aversive and/or harmful to insects, mechanistic sensitivity studies have been largely restricted to chordates. Furthermore, it is unknown if TRP-menthol sensing is as ancient as thermal and/or electrophile sensitivity. Combining genetic, optical, electrophysiological, behavioural and phylogenetic approaches, we tested the hypothesis that insect TRP channels play a conserved role in menthol sensing. We found that topical application of menthol to Drosophila melanogaster larvae elicits a Trpm- and TrpA1-dependent nocifensive rolling behaviour, which requires activation of Class IV nociceptor neurons. Further, in characterizing the evolution of TRP channels, we put forth the hypotheses that three previously undescribed TRPM channel clades (basal, αTRPM and ßTRPM), as well as TRPs with residues critical for menthol sensing, were present in ancestral bilaterians. This article is part of the Theo Murphy meeting issue 'Evolution of mechanisms and behaviour important for pain'.

Command or Obey? Homologous Neurons Differ in Hierarchical Position for the Generation of Homologous Behaviors.

In motor systems, higher-order neurons provide commands to lower-level central pattern generators (CPGs) that autonomously produce rhythmic motor patterns. Such hierarchical organization is often thought to be inherent in the anatomical position of the neurons. Here, however, we report that a neuron that is member of a CPG in one species acts as a higher-order neuron in another species. In the nudibranch mollusc, Melibe leonina, swim interneuron 1 (Si1) is in the CPG underlying swimming, firing rhythmic bursts of action potentials as part of the swim motor pattern. We found that its homolog in another nudibranch, Dendronotus iris, serves as a neuromodulatory command neuron for the CPG of a homologous swimming behavior. In Dendronotus, Si1 fired irregularly throughout the swim motor pattern. The burst and spike frequencies of Dendronotus swim CPG neurons correlated with Si1 firing frequency. Si1 activity was both necessary and sufficient for the initiation and maintenance of the swim motor pattern. Each Si1 was electrically coupled to all of the CPG neurons and made monosynaptic excitatory synapses with both Si3s. Si1 also bilaterally potentiated the excitatory synapse from Si3 to Si2. "Virtual neuromodulation" of both Si3-to-Si2 synapses using dynamic clamp combined with depolarization of both Si3s mimicked the effects of Si1 stimulation on the swim motor pattern. Thus, in Dendronotus, Si1 is a command neuron that turns on, maintains, and accelerates the motor pattern through synaptic and neuromodulatory actions, thereby differing from its homolog in Melibe in its functional position in the motor hierarchy.SIGNIFICANCE STATEMENT Cross-species comparisons of motor system organization can provide fundamental insights into their function and origin. Central pattern generators (CPGs) are lower in the functional hierarchy than the neurons that initiate and modulate their activity. This functional hierarchy is often reflected in neuroanatomical organization. This paper definitively shows that an identified cerebral ganglion neuron that is a member of a CPG underlying swimming in one nudibranch species serves as a command neuron for the same behavior in another species. We describe and test the synaptic and neuromodulatory mechanisms by which the command neuron initiates and accelerates rhythmic motor patterns. Thus, the functional position of neurons in a motor hierarchy can shift from one level to another over evolutionary time.

Artificial Synaptic Rewiring Demonstrates that Distinct Neural Circuit Configurations Underlie Homologous Behaviors.

Behavioral homology is often assumed to involve similarity in underlying neuronal mechanisms. Here, we provide a counterexample where homologous behaviors are produced by neurons with different synaptic connectivity. The nudibranch molluscs Melibe leonina and Dendronotus iris exhibit homologous swimming behaviors, consisting of alternating left and right body flexions. The swim central pattern generators (CPGs) in both species are composed of bilaterally symmetric interneurons, which are individually identified and reciprocally inhibit their contralateral counterparts, contributing to left-right burst alternation in the swim motor patterns. In Melibe, the swim CPG contains two parts that interact to produce stable rhythmic bursting; one part is the primary half-center kernel, and the other part, which consists of a bilateral pair of neurons called Si3, regulates period length. The Dendronotus swim CPG is simpler, with Si3 being part of the primary half-center oscillator. Application of curare (d-tubocurarine) selectively blocked the Si3 synapses in both species. In Melibe, curare application caused the burst duration of the swim motor pattern to lengthen, whereas in Dendronotus, curare halted bursting altogether. In both species, replacing the curare-blocked Si3 synapses with artificial synapses using dynamic clamp restored the original rhythmic bursting, thereby affirming the roles of those synapses. The curare-impaired bursting in Dendronotus was also restored by rewiring the homologous neurons into a Melibe-like primary half-center oscillator configuration, indicating that the connectivity itself could account for species differences in circuit responses to curare. The results suggest that synaptic connectivity diverged during evolution while behavior was conserved.

Variations on a theme: species differences in synaptic connectivity do not predict central pattern generator activity.

A fundamental question in comparative neuroethology is the extent to which synaptic wiring determines behavior vs. the extent to which it is constrained by phylogeny. We investigated this by examining the connectivity and activity of homologous neurons in different species. Melibe leonina and Dendronotus iris (Mollusca, Gastropoda, Nudibranchia) have homologous neurons and exhibit homologous swimming behaviors consisting of alternating left-right (LR) whole body flexions. Yet, a homologous interneuron (Si1) differs between the two species in its participation in the swim motor pattern (SMP) and synaptic connectivity. In this study we examined Si1 homologs in two additional nudibranchs: Flabellina iodinea, which evolved LR swimming independently of Melibe and Dendronotus, and Tritonia diomedea, which swims with dorsal-ventral (DV) body flexions. In Flabellina, the contralateral Si1s exhibit alternating rhythmic bursting activity during the SMP and are members of the swim central pattern generator (CPG), as in Melibe The Si1 homologs in Tritonia do not burst rhythmically during the DV SMP but are inhibited and receive bilaterally synchronous synaptic input. In both Flabellina and Tritonia, the Si1 homologs exhibit reciprocal inhibition, as in Melibe However, in Flabellina the inhibition is polysynaptic, whereas in Tritonia it is monosynaptic, as in Melibe In all species, the contralateral Si1s are electrically coupled. These results suggest that Flabellina and Melibe convergently evolved a swim CPG that contains Si1; however, they differ in monosynaptic connections. Connectivity is more similar between Tritonia and Melibe, which exhibit different swimming behaviors. Thus connectivity between homologous neurons varies independently of both behavior and phylogeny.NEW & NOTEWORTHY This research shows that the synaptic connectivity between homologous neurons exhibits species-specific variations on a basic theme. The neurons vary in the extent of electrical coupling and reciprocal inhibition. They also exhibit different patterns of activity during rhythmic motor behaviors that are not predicted by their circuitry. The circuitry does not map onto the phylogeny in a predictable fashion either. Thus neither neuronal homology nor species behavior is predictive of neural circuit connectivity.

Cortical Actin Alteration at the Matrix-Side Cytoplasm in Lung Adenocarcinoma Cells and Its Significance in Invasion.

OBJECTIVES: Cortical actin is a thin layer of filamentous (F-)actin that lies beneath the plasma membrane, and its role in pathophysiology remains unclear. We investigated the subcellular localization of cortical actin by the histopathological and experimental studies of lung adenocarcinomas. MATERIALS AND METHODS: The subcellular localization of cortical actin was studied in surgically resected lung adenocarcinomas tissues and in 3-dimensionally cultured lung adenocarcinoma A549 cells. RESULTS: In normal type II alveolar cells and the bronchiolar epithelium, cortical actin was localized to the apical-side cytoplasm. In invasive adenocarcinoma cells, cortical actin was frequently localized to the matrix side. The degree of cortical actin localized to the matrix side was associated with the loss of basement membrane and a poor prognosis. In A549 cell spheroids cultured in a type I collagen and basement membrane extract Matrigel™ mixed gel, cortical F-actin was localized to the matrix side with phosphorylated myosin light chain. Super-resolution and electron microscopy results suggest that compact wrinkling of the plasma membrane by myosin-mediated F-actin contraction is an explanation for cortical actin accumulation at the matrix side. The myosin II inhibitor blebbistatin suppressed the 3-dimensional collective migration of A549 cells induced by constitutively active Cdc42 and MT1-MMP. CONCLUSION: Cortical actin accumulation at the matrix-side cytoplasm of cancer cells occurs in invasive lung adenocarcinomas and it possibly participates in the migration of cancer cells through myosin-mediated contraction.