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A feature issue is being presented by a team of guest editors containing papers based on contributed submissions including studies presented at Optics and the Brain, held April 24-27, 2023 as part of Optica Biophotonics Congress: Optics in the Life Sciences, in Vancouver, Canada., Competing Interests: Optovue, Inc. (VJS)., (© 2024 Optica Publishing Group.)

Effective and precise mammalian transcriptome engineering technologies are needed to accelerate biological discovery and RNA therapeutics. Despite the promise of programmable CRISPR-Cas13 ribonucleases, their utility has been hampered by an incomplete understanding of guide RNA design rules and cellular toxicity resulting from off-target or collateral RNA cleavage. Here, we quantified the performance of over 127,000 RfxCas13d (CasRx) guide RNAs and systematically evaluated seven machine learning models to build a guide efficiency prediction algorithm orthogonally validated across multiple human cell types. Deep learning model interpretation revealed preferred sequence motifs and secondary features for highly efficient guides. We next identified and screened 46 novel Cas13d orthologs, finding that DjCas13d achieves low cellular toxicity and high specificity-even when targeting abundant transcripts in sensitive cell types, including stem cells and neurons. Our Cas13d guide efficiency model was successfully generalized to DjCas13d, illustrating the power of combining machine learning with ortholog discovery to advance RNA targeting in human cells., Competing Interests: Declaration of interests P.D.H. is a cofounder of Spotlight Therapeutics and Moment Biosciences and serves on their boards of directors and scientific advisory boards and is a scientific advisory board member to Arbor Biotechnologies, Vial Health, and Serotiny. P.D.H. and S.K. are inventors on patents relating to CRISPR technologies, including DjCas13d., (Copyright © 2023. Published by Elsevier Inc.)

We demonstrate a gradient refractive index (GRIN) microendoscope with an outer diameter of ∼1.2 mm and a length of ∼186 mm that can fit into a stereotactic surgical cannula. Two photon imaging at an excitation wavelength of 900 nm showed a field of view of ∼180 microns and a lateral and axial resolution of 0.86 microns and 9.6 microns respectively. The microendoscope was tested by imaging autofluorescence and second harmonic generation (SHG) in label-free human brain tissue. Furthermore, preliminary image analysis indicates that image classification models can predict if an image is from the subthalamic nucleus or the surrounding tissue using conventional, bench-top two-photon autofluorescence., Competing Interests: The authors declare that there are no conflicts of interest related to this article., (© 2023 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement.)

Background: In 2021, Canada implemented a pilot plasma program allowing some sexually active men who have sex with men (including but not limited to gay and bisexual men; gbMSM) to donate plasma. Changes to plasma donation policy could help address inequities in access to plasma donation and increase Canada's domestically collected plasma supply if more gbMSM donate as a result. We aimed to (1) examine views regarding plasma donation and the pilot program prior to implementation and (2) identify modifiable theory-informed predictors of gbMSM's intention to donate plasma., Methods: We developed, piloted, and disseminated a questionnaire informed by the Theoretical Domains Framework (TDF). We recruited gbMSM in London (ON) and Calgary (AB) to an anonymous, online cross-sectional survey., Results: A total of 246 gbMSM completed the survey. On scales from 1 (strongly disagree) to 5 (strongly agree), general intention to donate was high (mean = 4.24; SD = 0.94). The pilot program itself was mostly acceptable (mean = 3.71, SD = 1.16), but the intention to donate under the unique requirements of the pilot program was lower than general intention (mean = 3.58; SD = 1.26). Two domains from the theoretical domains framework (TDF) (beliefs about consequences of donating plasma and social influences) were independently associated with general intention to donate., Discussion: The pilot plasma program as an incremental step toward more inclusive policies was mostly viewed as acceptable by the impacted communities. Historical and ongoing exclusions create unique barriers to donation. There are clear opportunities for developing theory-informed interventions to support gbMSM to donate plasma as policies continue to become more inclusive and more become eligible to donate., (© 2023 The Authors. Transfusion published by Wiley Periodicals LLC on behalf of AABB.)

Background: Global chronic health worker shortages and stagnating routine immunization rates require new strategies to increase vaccination coverage and equity. As trained, trusted members of their local communities, community health workers (CHWs) are in a prime position to expand the immunization workforce and increase vaccination coverage in under-reached communities. Malawi is one of only a few countries that relies on CHWs-called Health Surveillance Assistants (HSAs) in Malawi-to administer routine immunizations, and as such offers a unique example of how this can be done., Case Presentation: We sought to describe the operational and programmatic characteristics of a functional CHW-led routine immunization program by conducting interviews with HSAs, HSA supervisors, ministry of health officials, and community members in Malawi. This case study describes how and where HSAs provide vaccinations, their vaccination-related responsibilities, training and supervision processes, vaccine safety considerations, and the community-level vaccine supply chain. Interview participants consistently described HSAs as a high-functioning vaccination cadre, skilled and dedicated to increasing vaccine access for children. They also noted a need to strengthen some aspects of professional support for HSAs, particularly related to training, supervision, and supply chain processes. Interviewees agreed that other countries should consider following Malawi's example and use CHWs to administer vaccines, provided they can be sufficiently trained and supported., Conclusions: This account from Malawi provides an example of how a CHW-led vaccination program operates. Leveraging CHWs as vaccinators is a promising yet under-explored task-shifting approach that shows potential to help countries maximize their health workforce, increase vaccination coverage and reach more zero-dose children. However, more research is needed to produce evidence on the impact of leveraging CHWs as vaccinators on patient safety, immunization coverage/vaccine equity, and cost-effectiveness as compared to use of other cadres for routine immunization., (© 2023. The Author(s).)

Optical sectioning structured illumination microscopy (OS-SIM) provides optical sectioning capability in wide-field microscopy. The required illumination patterns have traditionally been generated using spatial light modulators (SLM), laser interference patterns, or digital micromirror devices (DMDs) which are too complex to implement in miniscope systems. MicroLEDs have emerged as an alternative light source for patterned illumination due to their extreme brightness capability and small emitter sizes. This paper presents a directly addressable striped microLED microdisplay with 100 rows on a flexible cable (70 cm long) for use as an OS-SIM light source in a benchtop setup. The overall design of the microdisplay is described in detail with luminance-current-voltage characterization. OS-SIM implementation with a benchtop setup shows the optical sectioning capability of the system by imaging within a 500 µm thick fixed brain slice from a transgenic mouse where oligodendrocytes are labeled with a green fluorescent protein (GFP). Results show improved contrast in reconstructed optically sectioned images of 86.92% (OS-SIM) compared with 44.31% (pseudo-widefield). MicroLED based OS-SIM therefore offers a new capability for deep tissue widefield imaging.

Background: Canada has incrementally reduced restrictions to blood and plasma donation that impact men who have sex with men, gay, bisexual, and queer men, and some Two Spirit, transgender and non-binary individuals (MSM/2SGBTQ+). Prior to the launch of a pilot program in 2021 enabling some MSM/2SGBTQ + to donate source plasma, we explored the acceptability of the program among individuals who could become eligible to donate in the program., Methods: We invited men identifying as MSM/2SGBTQ + to participate in two consecutive semi-structured interviews to explore their views on blood and plasma donation policy, plasma donation, and the proposed Canadian plasma donation program. Interview transcripts were analyzed thematically and acceptability-related themes were mapped onto the Theoretical Framework of Acceptability., Results: Twenty-seven men identifying as having sex with men participated in 53 interviews. Eighteen themes were mapped onto the seven construct domains of the Theoretical Framework of Acceptability. Underlying all aspects of acceptability was a tension between four primary values influencing participants' views: altruism, equity, supply sufficiency, and evidence-based policy. The program was viewed as welcome progress on a discriminatory policy, with many excited to participate, yet tension with inequitable aspects of the program undermined support for the program and interest to contribute to it. The high demands of the program are unique for MSM/2SGBTQ + and are only tolerable as part of a program that is an incremental and instrumental step to more equitable donation policies., Conclusion: Findings highlight past experiences of exclusion in Canada as a unique and critical part of the context of the donation experience among MSM/2SGBTQ+. Despite the program's goals of greater inclusivity of MSM/2SGBTQ + individuals, the anticipated experience of the program included continued stigmatization and inequities. Future research should seek to understand the experienced views of MSM/2SGBTQ + donors to ensure that as policies change, policies are implemented equitably., (© 2023. The Author(s).)

Introduction: Community health workers (CHWs) could expand immunization access in under-reached communities by administering vaccines. This rapid review identifies countries where CHWs administered vaccines and synthesizes health systems factors that may contribute to or detract from the feasibility of CHWs administering vaccines., Methods: We conducted a rapid review of peer-reviewed literature from 3 databases and gray literature identified through web searches and by CHW subject matter experts. We treated extracted data on conditions related to vaccine administration by CHWs as qualitative data and conducted deductive content analysis., Results: We retained 32 documents from 497 initial records and identified 23 CHW cadres that vaccinated in 20 countries, ranging from long-established national programs delivering routine immunizations to pilot projects delivering 1 specific vaccine. CHWs who vaccinate face the following challenges: (1) inadequate supply chain training, (2) inadequate cold chain equipment, (3) transportation for supplies and to communities, (4) heavy existing workload, (5) inadequate or irregular remuneration, (6) inadequate or irregular supervision., Conclusion: To improve immunization coverage in underimmunized and zero-dose communities, countries where CHWs vaccinate should provide CHWs with adequate remuneration, supervision, supply chain support and management, and formal integration within the health system. CHWs administered vaccines in 20 of the 75 countries with documented CHW programs, suggesting the majority of an estimated 3.3 million CHWs globally do not yet administer vaccines. In light of health care workforce shortages and immunization equity gaps, further exacerbated by the COVID-19 pandemic, policymakers should consider task-shifting vaccine administration to CHWs to bolster immunization access for under-reached communities. Additional systematic documentation is needed to further explore best practices to support CHWs as vaccinators, especially related to supply chain, policy, safety, and efficacy., (© Gibson et al.)

The use of fluoride is effective in preventing dental caries. However, an excessive intake of fluoride leads to dental fluorosis, making it necessary to regularly monitor the fluoride intake especially for infants. There is hitherto a lack of information on fluoride content in infant foods from an Australian perspective. Therefore, this study aims to estimate the amount of fluoride content from a range of commercially available ready-to-eat (RTE) infant foods and drinks available in Australia. Based on an external calibration method, potentiometry involving a fluoride ion selective electrode and a silver|silver chloride reference electrode was conducted to analyse the fluoride content of a total of 326 solid food samples and 49 liquid food samples in this work. Our results showed an overall median (range) fluoride content of 0.16 (0.001-2.8) µg F/g of solid food samples, and 0.020 (0.002-1.2) µg F/mL of liquid food samples. In addition, ~77.5% of the liquid samples revealed a fluoride content < 0.05% µg F/mL. The highest variation of fluoride concentration (0.014-0.92 µg F/g) was found in formulas for ≥6 month-old infants. We have attributed the wide fluoride content variations in ready-to-eat infant foods and drinks to the processing steps, different ingredients and their origins, including water. In general, we found the fluoride content in most of the collected samples from Australian markets to be high and may therefore carry a risk of dental fluorosis. These results highlight the need for parents to receive appropriate information on the fluoride content of ready-to-eat infant food and drinks.

Apathy is a multi-dimensional syndrome associated with reduced initiation, executive function and emotion toward goal-directed behaviour. Affecting ∼30% of stroke patients, apathy can negatively impact rehabilitation outcomes and increase caregiver burden. However, relatively little is known about the multi-dimensional nature of post-stroke apathy and whether these dimensions map onto neuropsychological and neuroanatomical correlates. The present study aimed to address this question in a case series of stroke patients with apathy. 65 patients with acute stroke were assessed on a comprehensive battery of neuropsychological tasks and 12 patients were identified as having clinically significant apathy on one or more domains on the Dimensional Apathy Scale. Individual scores were compared to a group of healthy controls and normative data where available. Lesion mapping was completed from clinical CT and MRI scans to characterise the extent and locations of each patient's lesion. All participants performed significantly poorer than controls on one or more tasks. Difficulties with inhibition were observed across all dimensions. Prospective memory deficits were also common, while speed and social cognition were only reduced in initiation and emotional apathy, respectively. Verbal fluency was not impaired in any of the patients, despite previously established relationships with apathy. Lesions were predominantly located in right subcortical regions, with some additional frontal, temporal and cerebellar/brainstem involvement. There was substantial overlap in lesion locations within and between dimensions, such that similar apathy symptoms occurred in patients with very different lesion sites. Overall, our results suggest that neuropsychological and lesion profiles of apathy in stroke patients may be more complex and heterogenous than in neurodegenerative disease, possibly due to functional changes occurring beyond the lesion site., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Significance : Three-photon (3P) microscopy significantly increases the depth and resolution of in vivo imaging due to decreased scattering and nonlinear optical sectioning. Simultaneous excitation of multiple fluorescent proteins is essential to studying multicellular interactions and dynamics in the intact brain. Aim : We characterized the excitation laser pulses at a range of wavelengths for 3P microscopy, and then explored the application of tdTomato or mScarlet and EGFP for dual-color single-excitation structural 3P imaging deep in the living mouse brain. Approach : We used frequency-resolved optical gating to measure the spectral intensity, phase, and retrieved pulse widths at a range of wavelengths. Then, we performed in vivo single wavelength-excitation 3P imaging in the 1225- to 1360-nm range deep in the mouse cerebral cortex to evaluate the performance of tdTomato or mScarlet in combination with EGFP. Results : We find that tdTomato and mScarlet, expressed in oligodendrocytes and neurons respectively, have a high signal-to-background ratio in the 1300- to 1360-nm range, consistent with enhanced 3P cross-sections. Conclusions : These results suggest that a single excitation wavelength source is advantageous for multiple applications of dual-color brain imaging and highlight the importance of empirical characterization of individual fluorophores for 3P microscopy., (© 2022 The Authors.)

We present a high-resolution miniature, light-weight fluorescence microscope with electrowetting lens and onboard CMOS for high resolution volumetric imaging and structured illumination for rejection of out-of-focus and scattered light. The miniature microscope (SIMscope3D) delivers structured light using a coherent fiber bundle to obtain optical sectioning with an axial resolution of 18 µm. Volumetric imaging of eGFP labeled cells in fixed mouse brain tissue at depths up to 260 µm is demonstrated. The functionality of SIMscope3D to provide background free 3D imaging is shown by recording time series of microglia dynamics in awake mice at depths up to 120 µm in the brain., Competing Interests: The authors declare no conflicts of interest., (© 2022 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement.)

Raman spectroscopy using feature selection schemes has considerable advantages over gas chromatography for the analysis of fatty acids' composition changes. Here, we introduce an educational methodology to demonstrate the potential of micro-Raman spectroscopy to determine with high accuracy the unsaturation or saturation degrees and composition changes of the fatty acids found in the lipid droplets of the LNCaP prostate cancer cells that were treated with various fatty acids. The methodology uses highly discriminatory wavenumbers among fatty acids present in the sample selected by using the Support Vector Machine algorithm., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

We demonstrate a near-infrared, femtosecond, diode laser-based source with kW peak power for two-photon microscopy. At a wavelength of 976 nm, the system produces sub-ps pulses operating at a repetition rate of 10 MHz with kilowatt class peak powers suitable for deep tissue two-photon microscopy. The system, integrated with a laser-scanning microscope, images to a depth of 900 µm in a fixed sample of PLP-eGFP labeled mouse brain tissue. This represents a significant development that will lead to more efficient, compact, and accessible laser sources for biomedical imaging., Competing Interests: The authors declare no conflicts of interest., (© 2021 Optical Society of America under the terms of the OSA Open Access Publishing Agreement.)

Background: There is growing recognition of the need for fish to be better integrated into nutrition-sensitive strategies for addressing malnutrition. Fish are overwhelmingly produced by the small-scale sector, which supports food and nutrition security directly through the provision of fish and indirectly through the generation of income which can be used to purchase other desired foods. However, there has been relatively little research on the extent of food and nutrition security in specialised fishing communities. This study assessed food and nutrition security among households in specialised fishing communities in Komodo District, eastern Indonesia., Methods: We assessed the seasonal nutrition quality of household diets using the Food Consumption Score for nutritional analysis and food insecurity using the Household Food Insecurity Access Scale in 66 households across three communities, using a modified cluster sampling strategy. We calculated and generated descriptive statistics for these indicators with Microsoft Excel and ran a logistic generalized linear mixed model to determine factors associated with severe food insecurity using SPSS. We used semi-structured interviews and focus group discussions to understand perceptions of, change over time, and strategies for dealing with food shortfalls., Results: While most households have acceptable access to nutritious foods, especially protein and heme iron-rich foods, nearly one half of households consumed vitamin A rich foods on less than 3 days of the 7-day recall period in either season. More than half of households reported experiencing a moderate or severe level of food insecurity, with higher food insecurity in the wet season. Low maternal education (OR: 3.8, 95%CI 1.5-9.9) and lower household wealth (OR: 0.5, 95%CI 0.3-0.9) were found to be associated with a severe level of food insecurity. Household's consumptive and non-consumptive response strategies reflect adaptation to chronic food insecurity but are nutritionally and economically unsustainable., Conclusion: Households in specialised fishing communities in Komodo District consumed diets with low diversity and experienced high levels of food insecurity. There is a need for culturally-appropriate nutrition-sensitive strategies to enhance food and nutrition security in vulnerable fishing communities.

Small-scale coastal fisheries contribute directly and indirectly to the food and nutrition security of marine-dependent households. Fishers can apportion part of their catch for household consumption or use the income earned to purchase staples and other desired foods. Fish are an important animal-source food rich in micronutrients essential for cognitive development of children and for adult health, and a valuable addition to rice-based diets. Furthermore, the engagement of women in fisheries value chains and increased control over income may facilitate decision-making which improves nutrition outcomes for women and their children. Despite these contributions, food insecurity remains prevalent in many low and middle income fish-producing countries. This paper reports findings from an exploration of the interplaying factors leading to food and nutrition insecurity in three marine-dependent coastal communities in eastern Indonesia, focusing on the consumption pathway, that is, the contribution of fish to the diets and nutrition of women and children. The research was undertaken as a mixed-methods case study. The study found that over 50% of mother-child pairs failed to meet the minimum recommended dietary diversity, and, while fish was the main animal-source food in diets, the introduction of fish to infant and young child diets was delayed due to fears of allergies and illnesses. Moreover, access to nutrient-dense foods was affected by variable and insufficient income from fisheries-based livelihoods, isolation from markets, and the broader food environment. Given the shift towards 'nutrition-sensitive interventions' to improve the livelihoods and well-being of fisher households, these results highlight the need for analysis of the intra-household sharing of fish within fisher households, culturally-appropriate strategies to improve the quality of family and especially complementary foods, and efforts to increase physical access to nutrient-dense foods., Competing Interests: The authors have declared that no competing interests exist.

Calcineurin (CaN) phosphatase signaling is regulated by targeting CaN to substrates, inhibitors, and scaffold proteins containing docking motifs with the consensus sequence of PxIxIT. Here, we identify the docking of CaN to the γ isoform of MKK7, a component of the c-Jun N-terminal kinase (JNK) pathway. Because of alternative splicing of a single exon within the N-terminal domain, MKK7 γ encodes a unique PxIxIT motif (PIIVIT) that is not present in MKK7 α or β We found that MKK7 γ bound directly to CaN through this PIIVIT motif in vitro, immunoprecipitated with CaN from cell extracts, and exhibited fluorescence resonance energy transfer (FRET) with CaN in the cytoplasm but not in the nucleus of living cells. In contrast, MKK7 α and β exhibited no direct binding or FRET with CaN and were localized more in the nucleus than the cytoplasm. Furthermore, the inhibition of CaN phosphatase activity increased the basal phosphorylation of MKK7 γ but not MKK7 β Deletion of the MKK7 γ PIIVIT motif eliminated FRET with CaN and promoted MKK7 γ redistribution to the nucleus; however, the inhibition of CaN activity did not alter MKK7 γ localization, indicating that MKK7 γ cytoplasmic retention by CaN is phosphatase activity independent. Finally, the inhibition of CaN phosphatase activity in vascular smooth muscle cells, which express MKK7 γ mRNA, enhances JNK activation. Overall, we conclude that the MKK7 γ -specific PxIxIT motif promotes high-affinity CaN binding that could promote novel cross talk between CaN and JNK signaling by limiting MKK7 γ phosphorylation and restricting its localization to the cytoplasm., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

We present a miniature head mounted two-photon fiber-coupled microscope (2P-FCM) for neuronal imaging with active axial focusing enabled using a miniature electrowetting lens. We show three-dimensional two-photon imaging of neuronal structure and record neuronal activity from GCaMP6s fluorescence from multiple focal planes in a freely-moving mouse. Two-color simultaneous imaging of GFP and tdTomato fluorescence is also demonstrated. Additionally, dynamic control of the axial scanning of the electrowetting lens allows tilting of the focal plane enabling neurons in multiple depths to be imaged in a single plane. Two-photon imaging allows increased penetration depth in tissue yielding a working distance of 450 μm with an additional 180 μm of active axial focusing. The objective NA is 0.45 with a lateral resolution of 1.8 μm, an axial resolution of 10 μm, and a field-of-view of 240 μm diameter. The 2P-FCM has a weight of only ~2.5 g and is capable of repeatable and stable head-attachment. The 2P-FCM with dynamic axial scanning provides a new capability to record from functionally distinct neuronal layers, opening new opportunities in neuroscience research.

The repertoire and abundance of proteins displayed on the surface of neuronal dendrites are tuned by regulated fusion of recycling endosomes (REs) with the dendritic plasma membrane. While this process is critical for neuronal function and plasticity, how synaptic activity drives RE fusion remains unexplored. We demonstrate a multistep fusion mechanism that requires Ca 2+ from distinct sources. NMDA receptor Ca 2+ initiates RE fusion with the plasma membrane, while L-type voltage-gated Ca 2+ channels (L-VGCCs) regulate whether fused REs collapse into the membrane or reform without transferring their cargo to the cell surface. Accordingly, NMDA receptor activation triggered AMPA-type glutamate receptor trafficking to the dendritic surface in an L-VGCC-dependent manner. Conversely, potentiating L-VGCCs enhanced AMPA receptor surface expression only when NMDA receptors were also active. Thus L-VGCCs play a role in tuning activity-triggered surface expression of key synaptic proteins by gating the mode of RE fusion., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Determining the spatial relationship of individual proteins in dense assemblies remains a challenge for superresolution nanoscopy. The organization of aquaporin-4 (AQP4) into large plasma membrane assemblies provides an opportunity to image membrane-bound AQP4 antibodies (AQP4-IgG) and evaluate changes in their spatial distribution due to alterations in AQP4 isoform expression and AQP4-IgG epitope specificity. Using stimulated emission depletion nanoscopy, we imaged secondary antibody labeling of monoclonal AQP4-IgGs with differing epitope specificity bound to isolated tetramers (M1-AQP4) and large orthogonal arrays of AQP4 (M23-AQP4). Imaging secondary antibodies bound to M1-AQP4 allowed us to infer the size of individual AQP4-IgG binding events. This information was used to model the assembly of larger AQP4-IgG complexes on M23-AQP4 arrays. A scoring algorithm was generated from these models to characterize the spatial arrangement of bound AQP4-IgG antibodies, yielding multiple epitope-specific patterns of bound antibodies on M23-AQP4 arrays. Our results delineate an approach to infer spatial relationships within protein arrays using stimulated emission depletion nanoscopy, offering insight into how information on single antibody fluorescence events can be used to extract information from dense protein assemblies under a biologic context., (Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

AMPA receptors (AMPARs) are tetrameric ion channels assembled from GluA1-GluA4 subunits that mediate the majority of fast excitatory synaptic transmission in the brain. In the hippocampus, most synaptic AMPARs are composed of GluA1/2 or GluA2/3 with the GluA2 subunit preventing Ca(2+) influx. However, a small number of Ca(2+)-permeable GluA1 homomeric receptors reside in extrasynaptic locations where they can be rapidly recruited to synapses during synaptic plasticity. Phosphorylation of GluA1 S845 by the cAMP-dependent protein kinase (PKA) primes extrasynaptic receptors for synaptic insertion in response to NMDA receptor Ca(2+) signaling during long-term potentiation (LTP), while phosphatases dephosphorylate S845 and remove synaptic and extrasynaptic GluA1 during long-term depression (LTD). PKA and the Ca(2+)-activated phosphatase calcineurin (CaN) are targeted to GluA1 through binding to A-kinase anchoring protein 150 (AKAP150) in a complex with PSD-95, but we do not understand how the opposing activities of these enzymes are balanced to control plasticity. Here, we generated AKAP150ΔPIX knock-in mice to selectively disrupt CaN anchoring in vivo. We found that AKAP150ΔPIX mice lack LTD but express enhanced LTP at CA1 synapses. Accordingly, basal GluA1 S845 phosphorylation is elevated in AKAP150ΔPIX hippocampus, and LTD-induced dephosphorylation and removal of GluA1, AKAP150, and PSD-95 from synapses are impaired. In addition, basal synaptic activity of GluA2-lacking AMPARs is increased in AKAP150ΔPIX mice and pharmacologic antagonism of these receptors restores normal LTD and inhibits the enhanced LTP. Thus, AKAP150-anchored CaN opposes PKA phosphorylation of GluA1 to restrict synaptic incorporation of Ca(2+)-permeable AMPARs both basally and during LTP and LTD.

Purpose: To demonstrate the ability of multiphoton microscopy to obtain full three-dimensional high-resolution images of the intact mouse eye anterior chamber without need for enucleation., Methods: A custom multiphoton microscope was constructed and optimized for deep tissue imaging. Simultaneous two-photon autofluorescence (2PAF) and second harmonic generation (SHG) imaging were performed. A mouse holder and stereotaxic platform were designed to access different parts of the eye for imaging. A reservoir for keeping the eye moist was used during imaging sessions., Results: Non-invasive multiphoton images deep inside the anterior chamber of the mouse eye were obtained without the need for enucleation. The iris, corneal epithelium and endothelium, trabecular meshwork region and conjunctiva were visualized by the 2PAF and SHG signals. Identification of the anatomy was achieved by the intrinsic properties of the native tissue without any exogenous labeling. Images as deep as 600 microns into the eye were clearly demonstrated. Full three-dimensional image reconstructions of the entire anterior chamber were performed and analyzed using custom software., Conclusions: Multiphoton imaging is a highly promising tool for ophthalmic research. We have demonstrated the ability to image the entire anterior chamber of the mouse eye in its native state. These results provide a foundation for future in vivo studies of the eye.

Purpose: To image the human trabecular meshwork (TM) using a non-invasive, non-destructive technique without the application of exogenous label., Methods: Flat-mounted TM samples from a human cadaver eye were imaged using two nonlinear optical techniques: coherent anti-Stokes Raman scattering (CARS) and two-photon autofluorescence (TPAF). In TPAF, two optical photons are simultaneously absorbed and excite molecules in the sample that then emit a higher energy photon. The signal is predominately from collagen and elastin. The CARS technique uses two laser frequencies to specifically excite carbon-hydrogen bonds, allowing the visualization of lipid-rich cell membranes. Multiple images were taken along an axis perpendicular to the surface of the TM for subsequent analysis., Results: Analysis of multiple TPAF images of the TM reveals the characteristic overlapping bundles of collagen of various sizes. Simultaneous CARS imaging revealed elliptical structures of ~7×10 µm in diameter populating the meshwork which were consistent with TM cells. Irregularly shaped objects of ~4 µm diameter appeared in both the TPAF and CARS channels, and are consistent with melanin granules., Conclusions: CARS techniques were successful in imaging live TM cells in freshly isolated human TM samples. Similar images have been obtained with standard histological techniques, however the method described here has the advantage of being performed on unprocessed, unfixed tissue free from the potential distortions of the fine tissue morphology that can occur due to infusion of fixatives and treatment with alcohols. CARS imaging of the TM represents a new avenue for exploring details of aqueous outflow and TM cell physiology.

Purpose: To image the trabecular meshwork (TM) in its native unfixed state using a non-invasive, non-destructive technique., Methods: Two-photon microscopy (2PM), including two-photon excitation fluorescence (2PEF) and second harmonic generation (SHG), was used to image flat-mounted trabecular meshwork samples from human cadaver eyes. Multiple images were analyzed along the tissue axis (z-axis) to generate a three-dimensional (3D) model of the region., Results: A lattice of large collagen fibers (approximately 10 microm in diameter) were detected by inherent fluorescence (2PEF) and SHG. There are regions of both tightly overlapping bundles as well as fluid-filled regions visible from the surface of the TM. 3D analysis of multiple images reveals that the open regions deep in the TM penetrate the juxtacanalicular TM (JTM) and connect to the inner wall of Schlemm's canal (IWSC). These open regions may represent low-resistance fluid pathways between the anterior chamber and Schlemm's canal (SC)., Conclusions: 2PM imaging of the outflow system of the human eye documented collagenous structures solely from inherent optical properties, without addition of an exogenous fluorescent label. 2PM successfully imaged into the TM without the need for fixation, embedding, or histological processing. Deep penetration using advanced optical techniques revealed regions likely representing pores in the IWSC that have been documented by multiple electron microscope studies. Our work reveals that 2PM imaging has potential as a new metric for evaluating the aqueous outflow region of the human eye and is worthy of further exploration.

Triclosan (5-chloro-2-(2,4-dichlorophenoxy)phenol) is a potent antibacterial and antifungal compound that is widely used in personal care products, plastics, and fabrics. Recently triclosan has been shown to alter endocrine function in a variety of species. The purpose of this study was to determine effects of triclosan on pubertal development and thyroid hormone concentrations in the male rat. Weanling rats were exposed to 0, 3, 30, 100, 200, or 300 mg/kg of triclosan by oral gavage from postnatal day (PND) 23 to 53. Preputial separation (PPS) was examined beginning on PND 33. Rats were killed on PND 53, organ weights were recorded and serum was collected for subsequent analysis. Triclosan did not affect growth or the onset of PPS. Serum testosterone was significantly decreased at 200 mg/kg, however no effects were observed on androgen-dependent reproductive tissue weights. Triclosan significantly decreased total serum thyroxine (T4) in a dose-dependent manner at 30 mg/kg and higher (no observed effect level of 3 mg/kg). Triiodothyronine (T3) was significantly decreased only at 200 mg/kg, but thyroid stimulating hormone was not statistically different at any dose. Liver weights were significantly increased at 100 mg/kg triclosan and above suggesting that the induction of hepatic enzymes may have contributed to the altered T4 and T3 concentrations, but it does not appear to correlate with the T4 dose-response. This study demonstrates that triclosan exposure does not alter androgen-dependent tissue weights or onset of PPS; however, triclosan exposure significantly impacts thyroid hormone concentrations in the male juvenile rat.

Central to organization of signaling pathways are scaffolding, anchoring and adaptor proteins that mediate localized assembly of multi-protein complexes containing receptors, second messenger-generating enzymes, kinases, phosphatases, and substrates. At the postsynaptic density (PSD) of excitatory synapses, AMPA (AMPAR) and NMDA (NMDAR) glutamate receptors are linked to signaling proteins, the actin cytoskeleton, and synaptic adhesion molecules on dendritic spines through a network of scaffolding proteins that may play important roles regulating synaptic structure and receptor functions in synaptic plasticity underlying learning and memory. AMPARs are rapidly recruited to dendritic spines through NMDAR activation during induction of long-term potentiation (LTP) through pathways that also increase the size and F-actin content of spines. Phosphorylation of AMPAR-GluR1 subunits by the cAMP-dependent protein kinase (PKA) helps stabilize AMPARs recruited during LTP. In contrast, induction of long-term depression (LTD) leads to rapid calcineurin-protein phosphatase 2B (CaN) mediated dephosphorylation of PKA-phosphorylated GluR1 receptors, endocytic removal of AMPAR from synapses, and a reduction in spine size. However, mechanisms for coordinately regulating AMPAR localization, phosphorylation, and synaptic structure by PKA and CaN are not well understood. A kinase-anchoring protein (AKAP) 79/150 is a PKA- and CaN-anchoring protein that is linked to NMDARs and AMPARs through PSD-95 and SAP97 membrane-associated guanylate kinase (MAGUK) scaffolds. Importantly, disruption of PKA-anchoring in neurons and functional analysis of GluR1-MAGUK-AKAP79 complexes in heterologous cells suggests that AKAP79/150-anchored PKA and CaN may regulate AMPARs in LTD. In the work presented at the "First International Meeting on Anchored cAMP Signaling Pathways" (Berlin-Buch, Germany, October 15-16, 2005), we demonstrate that AKAP79/150 is targeted to dendritic spines by an N-terminal basic region that binds phosphatidylinositol-4,5-bisphosphate (PIP(2)), F-actin, and actin-linked cadherin adhesion molecules. Thus, anchoring of PKA and CaN as well as physical linkage of the AKAP to both cadherin-cytoskeletal and MAGUK-receptor complexes could play roles in coordinating changes in synaptic structure and receptor signaling functions underlying plasticity. Importantly, we provide evidence showing that NMDAR-CaN signaling pathways implicated in AMPAR regulation during LTD lead to a disruption of AKAP79/150 interactions with actin, MAGUKs, and cadherins and lead to a loss of the AKAP and anchored PKA from postsynapses. Our studies thus far indicate that this AKAP79/150 translocation depends on activation of CaN, F-actin reorganization, and possibly Ca(2+)-CaM binding to the N-terminal basic regions. Importantly, this tranlocation of the AKAP79/150-PKA complex from spines may shift the balance of PKA kinase and CaN/PP1 phosphatase activity at the postsynapse in favor of the phosphatases. This loss of PKA could then promote actions of CaN and PP1 during induction of LTD including maintaining AMPAR dephosphorylation, promoting AMPAR endocytosis, and preventing AMPAR recycling. Overall, these findings challenge the accepted notion that AKAPs are static anchors that position signaling proteins near fixed target substrates and instead suggest that AKAPs can function in more dynamic manners to regulate local signaling events.