A platform to deliver single and bi-specific Cas9/guide RNA to perturb genes in vitro and in vivo.

Although CRISPR-Cas9 technology is poised to revolutionize the treatment of diseases with underlying genetic mutations, it faces some significant issues limiting clinical entry. They include low-efficiency in vivo systemic delivery and undesired off-target effects. Here, we demonstrate, by modifying Cas9 with phosphorothioate-DNA oligos (PSs), that one can efficiently deliver single and bi-specific CRISPR-Cas9/guide RNA (gRNA) dimers in vitro and in vivo with reduced off-target effects. We show that PS-Cas9/gRNA-mediated gene knockout preserves chimeric antigen receptor T cell viability and expansion in vitro and in vivo. PS-Cas9/gRNA mediates gene perturbation in patient-derived tumor organoids and mouse xenograft tumors, leading to potent tumor antitumor effects. Further, HER2 antibody-PS-Cas9/gRNA conjugate selectively perturbs targeted genes in HER2+ ovarian cancer xenografts in vivo. Moreover, we created bi-specific PS-Cas9 with two gRNAs to target two adjacent sequences of the same gene, leading to efficient targeted gene disruption ex vivo and in vivo with markedly reduced unintended gene perturbation. Thus, the cell-penetrating PS-Cas9/gRNA can achieve efficient systemic delivery and precision in gene disruption.

The effect of food deserts on gynecologic cancer survival.

Objective: Living in a food desert is a known negative health risk, with recent literature finding an associated higher mortality in patients with cancers. Gynecologic cancers have not specifically been studied. We aimed to describe patients with gynecologic cancers who live in a food desert and determine if there is an association between living in a food desert and gynecologic cancer mortality. Methods: The 2013-2019 California Cancer Registry (CCR) was used to identify patients with endometrial, ovarian, or cervical cancers. Patient residential census tract was linked to food desert census tracts identified by the 2015 United States Department of Agriculture Food Access Research Atlas. Comorbidity data were obtained from the California Office of Statewide Health Planning and Development database (OSHPD). Treatment, diagnosis, and survival outcomes were obtained from the CCR's variables and compared by food desert status. Five-year disease-specific survival was analyzed by applying Cox proportional hazards analysis. Results: 40,340 gynecologic cancer cases were identified. 60.1 % had endometrial cancer, 23.2 % had ovarian cancer, and 15.9 % had cervical cancer. The average age of the cohort was 59.4 years, 48.0 % was non-Hispanic White, 50.3 % was privately insured, and 6.8 % of lived in a food desert. Living in a food desert was associated with higher disease-specific mortality for patients with gynecologic cancers (endometrial cancer HR 1.43p < 0.001 95 % CI 1.22-1.68; ovarian cancer HR 1.47p < 0.001 95 % CI 1.27-1.69; cervical cancer HR 1.24p = 0.045 95 % CI 1.01-1.54). Conclusion: Patients living in food deserts had worse disease-specific survival, making access to food a modifiable risk factor that may result in mitigating gynecologic cancer disparities.

Risk-reducing salpingo-oophorectomy among diverse patients with BRCA mutations at an urban public hospital: a mixed methods study.

OBJECTIVES: To assess the association of socioeconomic demographics with recommendation for and uptake of risk-reducing bilateral salpingo-oophorectomy (rrBSO) in patients with BRCA1 and BRCA2 (BRCA1/2) mutations. DESIGN: Retrospective cohort, semistructured qualitative interviews. SETTING AND PARTICIPANTS: BRCA1/2 mutation carriers at an urban, public hospital with a racially and socioeconomically diverse population. INTERVENTION: None. PRIMARY AND SECONDARY OUTCOMES: The primary outcomes were rate of rrBSO recommendation and completion. Secondary outcomes were sociodemographic variables associated with rrBSO completion. RESULTS: The cohort included 167 patients with BRCA1/2 mutations of whom 39% identified as black (n=65), 35% white (n=59) and 19% Hispanic (n=32). Over 95% (n=159) received the recommendation for age-appropriate rrBSO, and 52% (n=87) underwent rrBSO. Women who completed rrBSO were older in univariable analysis (p=0.05), but not in multivariable analysis. Completion of rrBSO was associated with residence in zip codes with lower unemployment and documented recommendation for rrBSO (p<0.05). All subjects who still received care in the health system (n=79) were invited to complete interviews regarding rrBSO decision-making, but only four completed surveys for a response rate of 5.1%. Themes that emerged included menopause, emotional impact and familial support. CONCLUSIONS: In this understudied population, genetic counselling and surrogates of financial health were associated with rrBSO uptake, highlighting genetics referrals and addressing social determinants of health as opportunities to improve cancer prevention and reduce health inequities. Our study demonstrates a need for more culturally centred recruiting methods for qualitative research in marginalised communities to ensure adequate representation in the literature regarding rrBSO.

Robotic HIPEC with use of a vaginal GelPoint®.

Patients with advanced stage ovarian cancers commonly undergo hyperthermic intraperitoneal chemotherapy (HIPEC) following interval debulking via exploratory laparotomy. This video demonstrates the feasibility of HIPEC delivery via a minimally invasive approach with the use of a vaginal GelPoint® port. This video demonstrates a 56-year-old patient with Stage 3 bilateral fallopian tube cancer who underwent 3 cycles of neoadjuvant chemotherapy with cisplatin and paclitaxel. Prior to administration of HIPEC the patient underwent an uncomplicated robotic assisted radical hysterectomy, bilateral salpingo-oopherectomy and infracolic omentectomy. Additionally, the falciform ligament was transected. The vaginal cuff was then used for placement of the GelPoint® port. The inflow and outflow cannulas were placed at the level of the liver and pelvis robotically. To minimize risk of inadvertent spillage, robotic obturators were replaced. Prior to administration of HIPEC, 4 L of warm saline was administered. An additional safety check was performed with no areas of leak. Cisplatin was administered for 90 min followed by sodium thiosulfate and 3 L of normal saline. Confirmation of no residual fluid was noted laparoscopically. The patient was discharged 2 days postoperatively without postoperative complications. In this video we demonstrated the innovative technique of performing HIPEC via a minimally invasive approach, that typically requires an open procedure. With the use of a vaginal Gelpoint® we were able to safely administer intraperitoneal chemotherapy without risk to our patient. We were also able to minimize their length of hospital stay and expedite postoperative recovery. Further implementation of this technique may improve hospital resource allocation.

Targeting PARG induces tumor cell growth inhibition and antitumor immune response by reducing phosphorylated STAT3 in ovarian cancer.

BACKGROUND: Ovarian cancer is the most lethal gynecological malignancy, with limited treatment options after failure of standard therapies. Despite the potential of poly(ADP-ribose) polymerase inhibitors in treating DNA damage response (DDR)-deficient ovarian cancer, the development of resistance and immunosuppression limit their efficacy, necessitating alternative therapeutic strategies. Inhibitors of poly(ADP-ribose) glycohydrolase (PARG) represent a novel class of inhibitors that are currently being assessed in preclinical and clinical studies for cancer treatment. METHODS: By using a PARG small-molecule inhibitor, COH34, and a cell-penetrating antibody targeting the PARG's catalytic domain, we investigated the effects of PARG inhibition on signal transducer and activator of transcription 3 (STAT3) in OVCAR8, PEO1, and Brca1-null ID8 ovarian cancer cell lines, as well as in immune cells. We examined PARG inhibition-induced effects on STAT3 phosphorylation, nuclear localization, target gene expression, and antitumor immune responses in vitro, in patient-derived tumor organoids, and in an immunocompetent Brca1-null ID8 ovarian mouse tumor model that mirrors DDR-deficient human high-grade serous ovarian cancer. We also tested the effects of overexpressing a constitutively activated STAT3 mutant on COH34-induced tumor cell growth inhibition. RESULTS: Our findings show that PARG inhibition downregulates STAT3 activity through dephosphorylation in ovarian cancer cells. Importantly, overexpression of a constitutively activated STAT3 mutant in tumor cells attenuates PARG inhibitor-induced growth inhibition. Additionally, PARG inhibition reduces STAT3 phosphorylation in immune cells, leading to the activation of antitumor immune responses, shown in immune cells cocultured with ovarian cancer patient tumor-derived organoids and in immune-competent mice-bearing mouse ovarian tumors. CONCLUSIONS: We have identified a novel antitumor mechanism underlying PARG inhibition beyond its primary antitumor effects through blocking DDR in ovarian cancer. Furthermore, targeting PARG activates antitumor immune responses, thereby potentially increasing response rates to immunotherapy in patients with ovarian cancer.

Impact of Sodium Thiosulfate on Prevention of Nephrotoxicities in HIPEC: An Ancillary Evaluation of Cisplatin-Induced Toxicities in Ovarian Cancer.

PURPOSE: Hyperthermic intraperitoneal chemotherapy (HIPEC) with cisplatin confers a survival benefit in epithelial ovarian cancer (EOC) but is associated with renal toxicity. Sodium thiosulfate (ST) is used for nephroprotection for HIPEC with cisplatin, but standard HIPEC practices vary. METHODS: A prospective, nonrandomized, clinical trial evaluated safety outcomes of HIPEC with cisplatin 75 mg/m2 during cytoreductive surgery (CRS) in patients with EOC (n = 34) and endometrial cancer (n = 6). Twenty-one patients received no ST (nST), and 19 received ST. Adverse events (AEs) were reported according to CTCAE v.5.0. Serum creatinine (Cr) was collected preoperatively and postoperatively (Days 5-8). Progression-free survival (PFS) was followed. Normal peritoneum was biopsied before and after HIPEC for whole transcriptomic sequencing to identify RNAseq signatures correlating with AEs. RESULTS: Forty patients had HIPEC at the time of interval or secondary CRS. Renal toxicities in the nST group were 33% any grade AE and 9% grade 3 AEs. The ST group demonstrated no renal AEs. Median postoperative Cr in the nST group was 1.1 mg/dL and 0.5 mg/dL in the ST group (p = 0.0001). Median change in Cr from preoperative to postoperative levels were + 53% (nST) compared with - 9.6% (ST) (p = 0.003). PFS did not differ between the ST and nST groups in primary or recurrent EOC patients. Renal AEs were associated with downregulation of metabolic pathways and upregulation of immune pathways. CONCLUSIONS: ST significantly reduces acute renal toxicity associated with HIPEC with cisplatin in ovarian cancer patients. As nephrotoxicity is high in HIPEC with cisplatin, nephroprotective agents should be considered.

Molecular logic of synaptic diversity between Drosophila tonic and phasic motoneurons.

Although neuronal subtypes display unique synaptic organization and function, the underlying transcriptional differences that establish these features are poorly understood. To identify molecular pathways that contribute to synaptic diversity, single-neuron Patch-seq RNA profiling was performed on Drosophila tonic and phasic glutamatergic motoneurons. Tonic motoneurons form weaker facilitating synapses onto single muscles, while phasic motoneurons form stronger depressing synapses onto multiple muscles. Super-resolution microscopy and in vivo imaging demonstrated that synaptic active zones in phasic motoneurons are more compact and display enhanced Ca2+ influx compared with their tonic counterparts. Genetic analysis identified unique synaptic properties that mapped onto gene expression differences for several cellular pathways, including distinct signaling ligands, post-translational modifications, and intracellular Ca2+ buffers. These findings provide insights into how unique transcriptomes drive functional and morphological differences between neuronal subtypes.

BRCA germline mutations in multiethnic gynecologic patients: A 10-year retrospective analysis from a single cancer institute.

Histologic and genetic mutation information from racially and ethnically diverse populations is warranted to better inform future cancer predisposition and promote health equity. A single institutional, retrospective capture of patients with gynecologic conditions and genetic susceptibilities to malignant neoplasms of the breast or ovaries was performed. This was achieved with manual curation of the electronic medical record (EMR) from 2010-2020 with the use of ICD-10 code searches. Among 8983 consecutive women identified with gynecologic conditions, 184 were diagnosed with pathogenic/likely pathogenic (P/LP) germline BRCA (gBRCA) mutations. Median age was 54 (22-90). Mutations included insertion/deletion (majority frameshift, 57.4%), substitution (32.4%), large structural rearrangement (5.4%), and alteration in splice site/intronic sequence (4.7%). A total of 48% were non-Hispanic White, 32% Hispanic or Latino, 13% Asian, 2% Black, and 5% Other. The most common pathology was high grade serous carcinoma (HGSC, 63%), followed by unclassified/high grade carcinoma (13%). Additional multigene panels led to the detection of 23 additional BRCA-positive patients with germline co-mutations and/or variants of uncertain significance in genes functionally involved in DNA repair mechanisms. Hispanic or Latino and Asian individuals comprised 45% of patients with concomitant gynecologic condition and gBRCA positivity in our cohort, confirming that germline mutations are represented across racial and ethnic groups. Insertion/deletion mutations, the majority of which led to a frameshift change, occurred in approximately half of our patient cohort, which may have prognostic implication for therapy resistance. Prospective studies are needed to unravel the significance of germline co-mutations in gynecologic patients.

A systematic review of artificial intelligence impact assessments.

Artificial intelligence (AI) is producing highly beneficial impacts in many domains, from transport to healthcare, from energy distribution to marketing, but it also raises concerns about undesirable ethical and social consequences. AI impact assessments (AI-IAs) are a way of identifying positive and negative impacts early on to safeguard AI's benefits and avoid its downsides. This article describes the first systematic review of these AI-IAs. Working with a population of 181 documents, the authors identified 38 actual AI-IAs and subjected them to a rigorous qualitative analysis with regard to their purpose, scope, organisational context, expected issues, timeframe, process and methods, transparency and challenges. The review demonstrates some convergence between AI-IAs. It also shows that the field is not yet at the point of full agreement on content, structure and implementation. The article suggests that AI-IAs are best understood as means to stimulate reflection and discussion concerning the social and ethical consequences of AI ecosystems. Based on the analysis of existing AI-IAs, the authors describe a baseline process of implementing AI-IAs that can be implemented by AI developers and vendors and that can be used as a critical yardstick by regulators and external observers to evaluate organisations' approaches to AI.

Tales of the unexpected.

A single event can completely change the direction of a career in science; four researchers share their stories.

Molecular Logic of Synaptic Diversity Between Drosophila Tonic and Phasic Motoneurons.

Although neuronal subtypes display unique synaptic organization and function, the underlying transcriptional differences that establish these features is poorly understood. To identify molecular pathways that contribute to synaptic diversity, single neuron PatchSeq RNA profiling was performed on Drosophila tonic and phasic glutamatergic motoneurons. Tonic motoneurons form weaker facilitating synapses onto single muscles, while phasic motoneurons form stronger depressing synapses onto multiple muscles. Super-resolution microscopy and in vivo imaging demonstrated synaptic active zones in phasic motoneurons are more compact and display enhanced Ca 2+ influx compared to their tonic counterparts. Genetic analysis identified unique synaptic properties that mapped onto gene expression differences for several cellular pathways, including distinct signaling ligands, post-translational modifications and intracellular Ca 2+ buffers. These findings provide insights into how unique transcriptomes drive functional and morphological differences between neuronal subtypes.

Niraparib-induced STAT3 inhibition increases its antitumor effects.

Recently, poly(ADP-ribosyl)ation polymerase inhibitors (PARPis), which induce synthetic lethality of tumor cells with DNA damage repair defects, have emerged as a promising therapy for ovarian, breast, and pancreatic cancer. Although the PARPi Olaparib is limited to treating cancer patients with DNA repair deficiencies, the PARPi Niraparib is FDA approved to treat ovarian cancer patients regardless of their status in DNA repair pathways. Despite differences in the affinity to PARP enzymes, the rationale behind the clinical use of Niraparib in patients without DNA repair deficiencies is still lacking. Moreover, only Olaparib has been approved for pancreatic ductal adenocarcinoma (PDAC) patients with BRCA mutations, accounting for only 5-7% of total PDACs. It remains unclear whether Niraparib could be beneficial to PDACs without BRCA mutations. We found that Niraparib inhibits ovarian and PDAC tumor cell growth, regardless of BRCA mutational status, more effectively than Olaparib. Unlike Olaparib, which is known to activate STAT3, Niraparib inhibits STAT3 activity in ovarian and PDAC cancer cell lines and patient tumors. Moreover, Niraparib regulates the expression of several STAT3 downstream genes involved in apoptosis. Overexpression of a constitutively activated STAT3 mutant rescues Niraparib-induced cancer cell apoptosis. Our results suggest that Niraparib inhibits pSTAT3 by interfering with SRC tyrosine kinase. Collectively, our studies provide a mechanism underlying Niraparib's ability to induce tumor cell apoptosis without BRCA mutations, suggesting the potential use of Niraparib for treating PDAC patients regardless of BRCA status.

The decoy SNARE Tomosyn sets tonic versus phasic release properties and is required for homeostatic synaptic plasticity.

Synaptic vesicle (SV) release probability (Pr) is a key presynaptic determinant of synaptic strength established by cell-intrinsic properties and further refined by plasticity. To characterize mechanisms that generate Pr heterogeneity between distinct neuronal populations, we examined glutamatergic tonic (Ib) and phasic (Is) motoneurons in Drosophila with stereotyped differences in Pr and synaptic plasticity. We found the decoy soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) Tomosyn is differentially expressed between these motoneuron subclasses and contributes to intrinsic differences in their synaptic output. Tomosyn expression enables tonic release in Ib motoneurons by reducing SNARE complex formation and suppressing Pr to generate decreased levels of SV fusion and enhanced resistance to synaptic fatigue. In contrast, phasic release dominates when Tomosyn expression is low, enabling high intrinsic Pr at Is terminals at the expense of sustained release and robust presynaptic potentiation. In addition, loss of Tomosyn disrupts the ability of tonic synapses to undergo presynaptic homeostatic potentiation.

Nerve cells transmit messages in the form of electrical and chemical signals. Electrical impulses travel along a neuron to the junction between two neighbouring cells, the synapse. There, chemical messengers called neurotransmitters are released from one cell and detected by the next, which can either excite or inhibit the recipient cell. Synapses differ in their ability to propagate signals and their signalling activity also fluctuates at times. Moreover, synaptic connections can be strengthened or weakened in a process called plasticity, which is a key part of learning new skills and recovering from a brain injury. It is thought that synaptic signalling might be amped up or dialled down to change the output of the connection between two cells, but exactly how this happens remains unclear. To investigate why synapses differ and how their signalling capabilities change, Sauvola et al. examined the connections between neurons and muscle cells in developing fruit flies. In fruit fly larvae, two types of neurons ­ called tonic Ib and phasic Is neurons ­ form synapses with muscle cells. But their synapses have different signalling properties: Ib synapses are weaker than Is synapses. Sauvola et al. hypothesised that a protein called Tomosyn ­ which is thought to restrict chemical signalling at the synapse ­ might be more active at weaker Ib synapses. Sauvola et al. found that Tomosyn was indeed more abundant at Ib synapses than at Is synapses, appearing to reflect their differences in signalling properties. In flies engineered to lack the Tomosyn protein, Ib synapses became four times stronger than usual, while Is synapses hardly changed. This supports the idea that Tomosyn restricts the release of neurotransmitters at typically weak Ib synapses. Further experiments showed Ib synapses in flies lacking Tomosyn also lost their malleability and ability to become strengthened during synaptic plasticity. Though the precise molecular interactions need further investigation, the findings suggest that Tomosyn is required for some forms of synaptic plasticity by controlling how much chemical signal neurons release. In summary, this work advances our understanding of synaptic signalling and brain plasticity, showing once again how the brain can change itself.

Habitability Models for Astrobiology.

Habitability has been generally defined as the capability of an environment to support life. Ecologists have been using Habitat Suitability Models (HSMs) for more than four decades to study the habitability of Earth from local to global scales. Astrobiologists have been proposing different habitability models for some time, with little integration and consistency among them, being different in function to those used by ecologists. Habitability models are not only used to determine whether environments are habitable, but they also are used to characterize what key factors are responsible for the gradual transition from low to high habitability states. Here we review and compare some of the different models used by ecologists and astrobiologists and suggest how they could be integrated into new habitability standards. Such standards will help improve the comparison and characterization of potentially habitable environments, prioritize target selections, and study correlations between habitability and biosignatures. Habitability models are the foundation of planetary habitability science, and the synergy between ecologists and astrobiologists is necessary to expand our understanding of the habitability of Earth, the Solar System, and extrasolar planets.

Synaptic Plasticity Induced by Differential Manipulation of Tonic and Phasic Motoneurons in Drosophila.

Structural and functional plasticity induced by neuronal competition is a common feature of developing nervous systems. However, the rules governing how postsynaptic cells differentiate between presynaptic inputs are unclear. In this study, we characterized synaptic interactions following manipulations of tonic Ib or phasic Is glutamatergic motoneurons that coinnervate postsynaptic muscles of male or female Drosophila melanogaster larvae. After identifying drivers for each neuronal subtype, we performed ablation or genetic manipulations to alter neuronal activity and examined the effects on synaptic innervation and function at neuromuscular junctions. Ablation of either Ib or Is resulted in decreased muscle response, with some functional compensation occurring in the Ib input when Is was missing. In contrast, the Is terminal failed to show functional or structural changes following loss of the coinnervating Ib input. Decreasing the activity of the Ib or Is neuron with tetanus toxin light chain resulted in structural changes in muscle innervation. Decreased Ib activity resulted in reduced active zone (AZ) number and decreased postsynaptic subsynaptic reticulum volume, with the emergence of filopodial-like protrusions from synaptic boutons of the Ib input. Decreased Is activity did not induce structural changes at its own synapses, but the coinnervating Ib motoneuron increased the number of synaptic boutons and AZs it formed. These findings indicate that tonic Ib and phasic Is motoneurons respond independently to changes in activity, with either functional or structural alterations in the Ib neuron occurring following ablation or reduced activity of the coinnervating Is input, respectively.SIGNIFICANCE STATEMENT Both invertebrate and vertebrate nervous systems display synaptic plasticity in response to behavioral experiences, indicating that underlying mechanisms emerged early in evolution. How specific neuronal classes innervating the same postsynaptic target display distinct types of plasticity is unclear. Here, we examined whether Drosophila tonic Ib and phasic Is motoneurons display competitive or cooperative interactions during innervation of the same muscle, or compensatory changes when the output of one motoneuron is altered. We established a system to differentially manipulate the motoneurons and examined the effects of cell type-specific changes to one of the inputs. Our findings indicate Ib and Is motoneurons respond differently to activity mismatch or loss of the coinnervating input, with the Ib subclass responding robustly compared with Is motoneurons.

Ovarian Cancer After Prophylactic Salpingectomy in a Patient With Germline BRCA1 Mutation.

BACKGROUND: Women with germline BRCA1 or BRCA2 mutations have a lifetime risk of ovarian cancer of up to 46%. Opportunistic salpingectomy has been advocated as a risk-reducing strategy owing to increasing recognition of tubal origin, yet evidence of efficacy in this high-risk population is limited. CASE: This is the case of a woman with a BRCA1 mutation who underwent prophylactic mastectomy and bilateral salpingectomy with ovarian retention before the age of 40 years. She did not undergo oophorectomy and subsequently developed stage IV high-grade serous ovarian cancer 4 years after her initial surgery. CONCLUSION: More research is needed to determine the role of prophylactic salpingectomy with delayed oophorectomy, optimal timing of completion oophorectomy, and the risks and benefits compared with up-front risk-reducing salpingo-oophorectomy.

Management and survival of patients with Mullerian adenosarcoma of the cervix without sarcomatous overgrowth desiring fertility preservation, a case report and review of the literature.

•Cervical Mullerian adenosarcoma is a tumor that affects reproductively aged women.•Hysterectomy had been the standard of care for these premenopausal women.•This case reports the most minimally invasive approach with no recurrence.•Accurate pathology interpretation is essential to diagnose and treat patients.•This is a rare tumor that if misdiagnosed or mischaracterized could be lethal.

Synaptic Properties and Plasticity Mechanisms of Invertebrate Tonic and Phasic Neurons.

Defining neuronal cell types and their associated biophysical and synaptic diversity has become an important goal in neuroscience as a mechanism to create comprehensive brain cell atlases in the post-genomic age. Beyond broad classification such as neurotransmitter expression, interneuron vs. pyramidal, sensory or motor, the field is still in the early stages of understanding closely related cell types. In both vertebrate and invertebrate nervous systems, one well-described distinction related to firing characteristics and synaptic release properties are tonic and phasic neuronal subtypes. In vertebrates, these classes were defined based on sustained firing responses during stimulation (tonic) vs. transient responses that rapidly adapt (phasic). In crustaceans, the distinction expanded to include synaptic release properties, with tonic motoneurons displaying sustained firing and weaker synapses that undergo short-term facilitation to maintain muscle contraction and posture. In contrast, phasic motoneurons with stronger synapses showed rapid depression and were recruited for short bursts during fast locomotion. Tonic and phasic motoneurons with similarities to those in crustaceans have been characterized in Drosophila, allowing the genetic toolkit associated with this model to be used for dissecting the unique properties and plasticity mechanisms for these neuronal subtypes. This review outlines general properties of invertebrate tonic and phasic motoneurons and highlights recent advances that characterize distinct synaptic and plasticity pathways associated with two closely related glutamatergic neuronal cell types that drive invertebrate locomotion.

Directional cerebrospinal fluid movement between brain ventricles in larval zebrafish.

BACKGROUND: Cerebrospinal fluid (CSF) contained within the brain ventricles contacts neuroepithelial progenitor cells during brain development. Dynamic properties of CSF movement may limit locally produced factors to specific regions of the developing brain. However, there is no study of in vivo CSF dynamics between ventricles in the embryonic brain. We address CSF movement using the zebrafish larva, during the major period of developmental neurogenesis. METHODS: CSF movement was monitored at two stages of zebrafish development: early larva [pharyngula stage; 27-30 h post-fertilization (hpf)] and late larva (hatching period; 51-54 hpf) using photoactivatable Kaede protein to calculate average maximum CSF velocity between ventricles. Potential roles for heartbeat in early CSF movement were investigated using tnnt2a mutant fish (tnnt2a (-/-)) and chemical [2,3 butanedione monoxime (BDM)] treatment. Cilia motility was monitored at these stages using the Tg(ßact:Arl13b-GFP) transgenic fish line. RESULTS: In wild-type early larva there is net CSF movement from the telencephalon to the combined diencephalic/mesencephalic superventricle. This movement directionality reverses at late larval stage. CSF moves directionally from diencephalic to rhombencephalic ventricles at both stages examined, with minimal movement from rhombencephalon to diencephalon. Directional movement is partially dependent on heartbeat, as indicated in assays of tnnt2a (-/-) fish and after BDM treatment. Brain cilia are immotile at the early larval stage. CONCLUSION: These data demonstrate directional movement of the embryonic CSF in the zebrafish model during the major period of developmental neurogenesis. A key conclusion is that CSF moves preferentially from the diencephalic into the rhombencephalic ventricle. In addition, the direction of CSF movement between telencephalic and diencephalic ventricles reverses between the early and late larval stages. CSF movement is partially dependent on heartbeat. At early larval stage, the absence of motile cilia indicates that cilia likely do not direct CSF movement. These data suggest that CSF components may be compartmentalized and could contribute to specialization of the early brain. In addition, CSF movement may also provide directional mechanical signaling.