Recessive variants in the intergenic NOS1AP-C1orf226 locus cause monogenic kidney disease responsive to anti-proteinuric treatment.

In genetic disease, an accurate expression landscape of disease genes and faithful animal models will enable precise genetic diagnoses and therapeutic discoveries, respectively. We previously discovered that variants in NOS1AP , encoding nitric oxide synthase 1 (NOS1) adaptor protein, cause monogenic nephrotic syndrome (NS). Here, we determined that an intergenic splice product of N OS1AP / Nos1ap and neighboring C1orf226/Gm7694 , which precludes NOS1 binding, is the predominant isoform in mammalian kidney transcriptional and proteomic data. Gm7694 -/- mice, whose allele exclusively disrupts the intergenic product, developed NS phenotypes. In two human NS subjects, we identified causative NOS1AP splice variants, including one predicted to abrogate intergenic splicing but initially misclassified as benign based on the canonical transcript. Finally, by modifying genetic background, we generated a faithful mouse model of NOS1AP -associated NS, which responded to anti-proteinuric treatment. This study highlights the importance of intergenic splicing and a potential treatment avenue in a mendelian disorder.

Gene drive and genetic sex conversion in the global agricultural pest Ceratitis capitata.

Homing-based gene drives are recently proposed interventions promising the area-wide, species-specific genetic control of harmful insect populations. Here we characterise a first set of gene drives in a tephritid agricultural pest species, the Mediterranean fruit fly Ceratitis capitata (medfly). Our results show that the medfly is highly amenable to homing-based gene drive strategies. By targeting the medfly transformer gene, we also demonstrate how CRISPR-Cas9 gene drive can be coupled to sex conversion, whereby genetic females are transformed into fertile and harmless XX males. Given this unique malleability of sex determination, we modelled gene drive interventions that couple sex conversion and female sterility and found that such approaches could be effective and tolerant of resistant allele selection in the target population. Our results open the door for developing gene drive strains for the population suppression of the medfly and related tephritid pests by co-targeting female reproduction and shifting the reproductive sex ratio towards males. They demonstrate the untapped potential for gene drives to tackle agricultural pests in an environmentally friendly and economical way.

Regenerative capacity of neural tissue scales with changes in tissue mechanics post injury.

Spinal cord injuries have devastating consequences for humans, as mammalian neurons of the central nervous system (CNS) cannot regenerate. In the peripheral nervous system (PNS), however, neurons may regenerate to restore lost function following injury. While mammalian CNS tissue softens after injury, how PNS tissue mechanics changes in response to mechanical trauma is currently poorly understood. Here we characterised mechanical rat nerve tissue properties before and after in vivo crush and transection injuries using atomic force microscopy-based indentation measurements. Unlike CNS tissue, PNS tissue significantly stiffened after both types of tissue damage. This nerve tissue stiffening strongly correlated with an increase in collagen I levels. Schwann cells, which crucially support PNS regeneration, became more motile and proliferative on stiffer substrates in vitro, suggesting that changes in tissue stiffness may play a key role in facilitating or impeding nervous system regeneration.

A large-scale, targeted metabolomics method for the analysis and quantification of metabolites in human plasma via liquid chromatography-mass spectrometry.

Metabolomics is the study of small molecules, primarily metabolites, that are produced during metabolic processes. Analysis of the composition of an organism's metabolome can yield useful information about an individual's health status at any given time. In recent years, the development of large-scale, targeted metabolomic methods has allowed for the analysis of biological samples using analytical techniques such as LC-MS/MS. This paper presents a large-scale metabolomics method for analysis of biological samples, with a focus on quantification of metabolites found in blood plasma. The method comprises a 10-min chromatographic separation using HILIC and RP stationary phases combined with positive and negative electrospray ionization in order to maximize metabolome coverage. Complete analysis of a single sample can be achieved in as little as 40 min using the two columns and dual modes of ionization. With 540 metabolites and the inclusion of over 200 analytical standards, this method is comprehensive and quantitatively robust when compared to current targeted metabolomics methods. This study uses a large-scale evaluation of metabolite recovery from plasma that enables absolute quantification of metabolites by correcting for analyte loss throughout processes such as extraction, handling, or storage. In addition, the method was applied to plasma collected from adjuvant breast cancer patients to confirm the suitability of the method to clinical samples.

Integrin-Driven Axon Regeneration in the Spinal Cord Activates a Distinctive CNS Regeneration Program.

The peripheral branch of sensory dorsal root ganglion (DRG) neurons regenerates readily after injury unlike their central branch in the spinal cord. However, extensive regeneration and reconnection of sensory axons in the spinal cord can be driven by the expression of α9 integrin and its activator kindlin-1 (α9k1), which enable axons to interact with tenascin-C. To elucidate the mechanisms and downstream pathways affected by activated integrin expression and central regeneration, we conducted transcriptomic analyses of adult male rat DRG sensory neurons transduced with α9k1, and controls, with and without axotomy of the central branch. Expression of α9k1 without the central axotomy led to upregulation of a known PNS regeneration program, including many genes associated with peripheral nerve regeneration. Coupling α9k1 treatment with dorsal root axotomy led to extensive central axonal regeneration. In addition to the program upregulated by α9k1 expression, regeneration in the spinal cord led to expression of a distinctive CNS regeneration program, including genes associated with ubiquitination, autophagy, endoplasmic reticulum (ER), trafficking, and signaling. Pharmacological inhibition of these processes blocked the regeneration of axons from DRGs and human iPSC-derived sensory neurons, validating their causal contributions to sensory regeneration. This CNS regeneration-associated program showed little correlation with either embryonic development or PNS regeneration programs. Potential transcriptional drivers of this CNS program coupled to regeneration include Mef2a, Runx3, E2f4, and Yy1. Signaling from integrins primes sensory neurons for regeneration, but their axon growth in the CNS is associated with an additional distinctive program that differs from that involved in PNS regeneration.SIGNIFICANCE STATEMENT Restoration of neurologic function after spinal cord injury has yet to be achieved in human patients. To accomplish this, severed nerve fibers must be made to regenerate. Reconstruction of nerve pathways has not been possible, but recently, a method for stimulating long-distance axon regeneration of sensory fibers in rodents has been developed. This research uses profiling of messenger RNAs in the regenerating sensory neurons to discover which mechanisms are activated. This study shows that the regenerating neurons initiate a novel CNS regeneration program which includes molecular transport, autophagy, ubiquitination, and modulation of the endoplasmic reticulum (ER). The study identifies mechanisms that neurons need to activate to regenerate their nerve fibers.

Body size dictates physiological and behavioural responses to hypoxia and elevated water temperatures in Murray cod (Maccullochella peelii).

Increasing drought frequency and duration pose a significant threat to fish species in dryland river systems. As ectotherms, fish thermal and hypoxia tolerances directly determine the capacity of species to persist in these environments during low flow periods when water temperatures are high and waterbodies become highly stratified. Chronic thermal stress can compound the impacts of acute hypoxic events on fish resulting in significant fish mortality; however, it is not known if all size classes are equally susceptible, or if the allometric scaling of physiological processes means some size classes are disproportionately affected. We investigated the physiological responses of Murray cod (Maccullochella peelii) over a four-fold body size range (0.2-3000 g) to acute changes in water temperature and oxygen concentration following 4 weeks of acclimation to representative spring (20°C) and summer (28°C) water temperatures. We recorded maximum thermal tolerance (CT max), oxygen limited thermal tolerance (PCTmax ), lowest tolerable oxygen level (as the oxygen level at which lose equilibrium; O2,LOE), gill ventilation rates and aerial surface respiration threshold, blood oxygen transport capacity and lactate accumulation. Acclimation to elevated water temperatures improved thermal and hypoxia tolerance metrics across all size classes. However, body size significantly affected thermal and hypoxia responses. Small M. peelii were significantly less hypoxia tolerant than larger individuals, while larger fish were significantly less thermal tolerant than smaller fish. Hypoxia constrained thermal tolerance in M. peelii, with both small and large fish disproportionally compromised relative to mid-sized fish. Our findings indicate that both very small/young (larvae, fry, fingerlings) and very large/older M. peelii in dryland rivers are at significant risk from the combined impacts of a warming and drying climate and water extraction. These data will inform policy decisions that serve to balance competing demands on precious freshwater resources.

Improving adeno-associated viral (AAV) vector-mediated transgene expression in retinal ganglion cells: comparison of five promoters.

Recombinant adeno-associated viral vectors (AAVs) are an effective system for gene transfer. AAV serotype 2 (AAV2) is commonly used to deliver transgenes to retinal ganglion cells (RGCs) via intravitreal injection. The AAV serotype however is not the only factor contributing to the effectiveness of gene therapies. Promoters influence the strength and cell-selectivity of transgene expression. This study compares five promoters designed to maximise AAV2 cargo space for gene delivery: chicken ß-actin (CBA), cytomegalovirus (CMV), short CMV early enhancer/chicken ß-actin/short ß-globulin intron (sCAG), mouse phosphoglycerate kinase (PGK), and human synapsin (SYN). The promoters driving enhanced green fluorescent protein (eGFP) were examined in adult C57BL/6J mice eyes and tissues of the visual system. eGFP expression was strongest in the retina, optic nerves and brain when driven by the sCAG and SYN promoters. CBA, CMV, and PGK had moderate expression by comparison. The SYN promoter had almost exclusive transgene expression in RGCs. The PGK promoter had predominant expression in both RGCs and AII amacrine cells. The ubiquitous CBA, CMV, and sCAG promoters expressed eGFP in a variety of cell types across multiple retinal layers including Müller glia and astrocytes. We also found that these promoters could transduce human retina ex vivo, although expression was predominantly in glial cells due to low RGC viability. Taken together, this promoter comparison study contributes to optimising AAV-mediated transduction in the retina, and could be valuable for research in ocular disorders, particularly those with large or complex genetic cargos.

NCK1 Modulates Neuronal Actin Dynamics and Promotes Dendritic Spine, Synapse, and Memory Formation.

Memory formation and maintenance is a dynamic process involving the modulation of the actin cytoskeleton at synapses. Understanding the signaling pathways that contribute to actin modulation is important for our understanding of synapse formation and function, as well as learning and memory. Here, we focused on the importance of the actin regulator, noncatalytic region of tyrosine kinase adaptor protein 1 (NCK1), in hippocampal dependent behaviors and development. We report that male mice lacking NCK1 have impairments in both short-term and working memory, as well as spatial learning. Additionally, we report sex differences in memory impairment showing that female mice deficient in NCK1 fail at reversal learning in a spatial learning task. We find that NCK1 is expressed in postmitotic neurons but is dispensable for neuronal proliferation and migration in the developing hippocampus. Morphologically, NCK1 is not necessary for overall neuronal dendrite development. However, neurons lacking NCK1 have lower dendritic spine and synapse densities in vitro and in vivo EM analysis reveal increased postsynaptic density (PSD) thickness in the hippocampal CA1 region of NCK1-deficient mice. Mechanistically, we find the turnover of actin-filaments in dendritic spines is accelerated in neurons that lack NCK1. Together, these findings suggest that NCK1 contributes to hippocampal-dependent memory by stabilizing actin dynamics and dendritic spine formation.SIGNIFICANCE STATEMENT Understanding the molecular signaling pathways that contribute to memory formation, maintenance, and elimination will lead to a better understanding of the genetic influences on cognition and cognitive disorders and will direct future therapeutics. Here, we report that the noncatalytic region of tyrosine kinase adaptor protein 1 (NCK1) adaptor protein modulates actin-filament turnover in hippocampal dendritic spines. Mice lacking NCK1 show sex-dependent deficits in hippocampal memory formation tasks, have altered postsynaptic densities, and reduced synaptic density. Together, our work implicates NCK1 in the regulation of actin cytoskeleton dynamics and normal synapse development which is essential for memory formation.

Perineuronal nets affect memory and learning after synapse withdrawal.

Perineuronal nets (PNNs) enwrap mature neurons, playing a role in the control of plasticity and synapse dynamics. PNNs have been shown to have effects on memory formation, retention and extinction in a variety of animal models. It has been proposed that the cavities in PNNs, which contain synapses, can act as a memory store and that they remain stable after events that cause synaptic withdrawal such as anoxia or hibernation. We examine this idea by monitoring place memory before and after synaptic withdrawal caused by acute hibernation-like state (HLS). Animals lacking hippocampal PNNs due to enzymatic digestion by chondroitinase ABC or knockout of the PNN component aggrecan were compared with wild type controls. HLS-induced synapse withdrawal caused a memory deficit, but not to the level of untreated naïve animals and not worsened by PNN attenuation. After HLS, only animals lacking PNNs showed memory restoration or relearning. Absence of PNNs affected the restoration of excitatory synapses on PNN-bearing neurons. The results support a role for hippocampal PNNs in learning, but not in long-term memory storage for correction of deficits.

Phosphite synthetic auxotrophy as an effective biocontainment strategy for the industrial chassis Pseudomonas putida.

The inclusion of biosafety strategies into strain engineering pipelines is crucial for safe-by-design biobased processes. This in turn might enable a more rapid regulatory acceptance of bioengineered organisms in both industrial and environmental applications. For this reason, we equipped the industrially relevant microbial chassis Pseudomonas putida KT2440 with an effective biocontainment strategy based on a synthetic dependency on phosphite, which is generally not readily available in the environment. The produced PSAG-9 strain was first engineered to assimilate phosphite through the genome-integration of a phosphite dehydrogenase and a phosphite-specific transport complex. Subsequently, to deter the strain from growing on naturally assimilated phosphate, all native genes related to its transport were identified and deleted generating a strain unable to grow on media containing any phosphorous source other than phosphite. PSAG-9 exhibited fitness levels with phosphite similar to those of the wild type with phosphate, and low levels of escape frequency. Beyond biosafety, this strategy endowed P. putida with the capacity to be cultured under non-sterile conditions using phosphite as the sole phosphorous source with a reduced risk of contamination by other microbes, while displaying enhanced NADH regenerative capacity. These industrially beneficial features complement the metabolic advantages for which this species is known for, thereby strengthening it as a synthetic biology chassis with potential uses in industry, with suitability towards environmental release.

The extracellular matrix and perineuronal nets in memory.

All components of the CNS are surrounded by a diffuse extracellular matrix (ECM) containing chondroitin sulphate proteoglycans (CSPGs), heparan sulphate proteoglycans (HSPGs), hyaluronan, various glycoproteins including tenascins and thrombospondin, and many other molecules that are secreted into the ECM and bind to ECM components. In addition, some neurons, particularly inhibitory GABAergic parvalbumin-positive (PV) interneurons, are surrounded by a more condensed cartilage-like ECM called perineuronal nets (PNNs). PNNs surround the soma and proximal dendrites as net-like structures that surround the synapses. Attention has focused on the role of PNNs in the control of plasticity, but it is now clear that PNNs also play an important part in the modulation of memory. In this review we summarize the role of the ECM, particularly the PNNs, in the control of various types of memory and their participation in memory pathology. PNNs are now being considered as a target for the treatment of impaired memory. There are many potential treatment targets in PNNs, mainly through modulation of the sulphation, binding, and production of the various CSPGs that they contain or through digestion of their sulphated glycosaminoglycans.

Proteoglycan Sulphation in the Function of the Mature Central Nervous System.

Chondroitin sulphate and heparan sulphate proteoglycans (CSPGS and HSPGs) are found throughout the central nervous system (CNS). CSPGs are ubiquitous in the diffuse extracellular matrix (ECM) between cells and are a major component of perineuronal nets (PNNs), the condensed ECM present around some neurons. HSPGs are more associated with the surface of neurons and glia, with synapses and in the PNNs. Both CSPGs and HSPGs consist of a protein core to which are attached repeating disaccharide chains modified by sulphation at various positions. The sequence of sulphation gives the chains a unique structure and local charge density. These sulphation codes govern the binding properties and biological effects of the proteoglycans. CSPGs are sulphated along their length, the main forms being 6- and 4-sulphated. In general, the chondroitin 4-sulphates are inhibitory to cell attachment and migration, while chondroitin 6-sulphates are more permissive. HSPGs tend to be sulphated in isolated motifs with un-sulphated regions in between. The sulphation patterns of HS motifs and of CS glycan chains govern their binding to the PTPsigma receptor and binding of many effector molecules to the proteoglycans, such as growth factors, morphogens, and molecules involved in neurodegenerative disease. Sulphation patterns change as a result of injury, inflammation and ageing. For CSPGs, attention has focussed on PNNs and their role in the control of plasticity and memory, and on the soluble CSPGs upregulated in glial scar tissue that can inhibit axon regeneration. HSPGs have key roles in development, regulating cell migration and axon growth. In the adult CNS, they have been associated with tau aggregation and amyloid-beta processing, synaptogenesis, growth factor signalling and as a component of the stem cell niche. These functions of CSPGs and HSPGs are strongly influenced by the pattern of sulphation of the glycan chains, the sulphation code. This review focuses on these sulphation patterns and their effects on the function of the mature CNS.

Microglia-mediated degradation of perineuronal nets promotes pain.

Activation of microglia in the spinal cord dorsal horn after peripheral nerve injury contributes to the development of pain hypersensitivity. How activated microglia selectively enhance the activity of spinal nociceptive circuits is not well understood. We discovered that after peripheral nerve injury, microglia degrade extracellular matrix structures, perineuronal nets (PNNs), in lamina I of the spinal cord dorsal horn. Lamina I PNNs selectively enwrap spinoparabrachial projection neurons, which integrate nociceptive information in the spinal cord and convey it to supraspinal brain regions to induce pain sensation. Degradation of PNNs by microglia enhances the activity of projection neurons and induces pain-related behaviors. Thus, nerve injury-induced degradation of PNNs is a mechanism by which microglia selectively augment the output of spinal nociceptive circuits and cause pain hypersensitivity.

Mllt11 Regulates Migration and Neurite Outgrowth of Cortical Projection Neurons during Development.

The formation of connections within the mammalian neocortex is highly regulated by both extracellular guidance mechanisms and intrinsic gene expression programs. There are two types of cortical projection neurons (CPNs): those that project locally and interhemispherically and those that project to subcerebral structures such as the thalamus, hindbrain, and spinal cord. The regulation of cortical projection morphologies is not yet fully understood at the molecular level. Here, we report a role for Mllt11 (Myeloid/lymphoid or mixed-lineage leukemia; translocated to chromosome 11/All1 Fused Gene From Chromosome 1q) in the migration and neurite outgrowth of callosal projection neurons during mouse brain formation. We show that Mllt11 expression is exclusive to developing neurons and is enriched in the developing cortical plate (CP) during the formation of the superficial cortical layers. In cultured primary cortical neurons, Mllt11 is detected in varicosities and growth cones as well as the soma. Using conditional loss-of-function and gain-of-function analysis we show that Mllt11 is required for neuritogenesis and proper migration of upper layer CPNs. Loss of Mllt11 in the superficial cortex of male and female neonates leads to a severe reduction in fibers crossing the corpus callosum (CC), a progressive loss in the maintenance of upper layer projection neuron gene expression, and reduced complexity of dendritic arborization. Proteomic analysis revealed that Mllt11 associates with stabilized microtubules, and Mllt11 loss affected microtubule staining in callosal axons. Taken together, our findings support a role for Mllt11 in promoting the formation of mature upper-layer neuron morphologies and connectivity in the cerebral cortex.SIGNIFICANCE STATEMENT The regulation of cortical projection neuron (CPN) morphologies is an area of active investigation since the time of Cajal. Yet the molecular mechanisms of how the complex dendritic and axonal morphologies of projection neurons are formed remains incompletely understood. Although conditional mutagenesis analysis in the mouse, coupled with overexpression assays in the developing fetal brain, we show that a novel protein called Mllt11 is sufficient and necessary to regulate the dendritic and axonal characteristics of callosal projection neurons in the developing mammalian neocortex. Furthermore, we show that Mllt11 interacts with microtubules, likely accounting for its role in neuritogenesis.

Prevention of the foreign body response to implantable medical devices by inflammasome inhibition.

SignificanceImplantable electronic medical devices (IEMDs) are used for some clinical applications, representing an exciting prospect for the transformative treatment of intractable conditions such Parkinson's disease, deafness, and paralysis. The use of IEMDs is limited at the moment because, over time, a foreign body reaction (FBR) develops at the device-neural interface such that ultimately the IEMD fails and needs to be removed. Here, we show that macrophage nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activity drives the FBR in a nerve injury model yet integration of an NLRP3 inhibitor into the device prevents FBR while allowing full healing of damaged neural tissue to occur.

Long-Term Cultures of Spinal Cord Interneurons.

Spinal cord interneurons (SpINs) are highly diverse population of neurons that play a significant role in circuit reorganization and spontaneous recovery after spinal cord injury. Regeneration of SpIN axons across rodent spinal injuries has been demonstrated after modification of the environment and neurotrophin treatment, but development of methods to enhance the intrinsic regenerative ability of SpINs is needed. There is a lack of described in vitro models of spinal cord neurons in which to develop new regeneration treatments. For this reason, we developed a new model of mouse primary spinal cord neuronal culture in which to analyze maturation, morphology, physiology, connectivity and regeneration of identified interneurons. Isolated from E14 mice, the neurons mature over 15 days in vitro, demonstrated by expression of maturity markers, electrophysiological patch-clamp recordings, and formation of synapses. The neurons express markers of SpINs, including Tlx3, Lmx1b, Lbx1, Chx10, and Pax2. The neurons demonstrate distinct morphologies and some form perineuronal nets in long-term cultivation. Live neurons in various maturation stages were axotomized, using a 900 nm multiphoton laser and their fate was observed overnight. The percentage of axons that regenerated declined with neuronal maturity. This model of SpINs will be a valuable tool in future regenerative, developmental, and functional studies alongside existing models using cortical or hippocampal neurons.

Heterozygous Dcc Mutant Mice Have a Subtle Locomotor Phenotype.

Axon guidance receptors such as deleted in colorectal cancer (DCC) contribute to the normal formation of neural circuits, and their mutations can be associated with neural defects. In humans, heterozygous mutations in DCC have been linked to congenital mirror movements, which are involuntary movements on one side of the body that mirror voluntary movements of the opposite side. In mice, obvious hopping phenotypes have been reported for bi-allelic Dcc mutations, while heterozygous mutants have not been closely examined. We hypothesized that a detailed characterization of Dcc heterozygous mice may reveal impaired corticospinal and spinal functions. Anterograde tracing of the Dcc+/- motor cortex revealed a normally projecting corticospinal tract, intracortical microstimulation (ICMS) evoked normal contralateral motor responses, and behavioral tests showed normal skilled forelimb coordination. Gait analyses also showed a normal locomotor pattern and rhythm in adult Dcc+/- mice during treadmill locomotion, except for a decreased occurrence of out-of-phase walk and an increased duty cycle of the stance phase at slow walking speed. Neonatal isolated Dcc+/- spinal cords had normal left-right and flexor-extensor coupling, along with normal locomotor pattern and rhythm, except for an increase in the flexor-related motoneuronal output. Although Dcc+/- mice do not exhibit any obvious bilateral impairments like those in humans, they exhibit subtle motor deficits during neonatal and adult locomotion.

Oral treatment of 4-methylumbelliferone reduced perineuronal nets and improved recognition memory in mice.

Hyaluronan (HA) is a core constituent of perineuronal nets (PNNs) that surround subpopulations of neurones. The PNNs control synaptic stabilization in both the developing and adult central nervous system, and disruption of PNNs has shown to reactivate neuroplasticity. We investigated the possibility of memory prolongation by attenuating PNN formation using 4-methylumbelliferone (4-MU), an inhibitor of HA synthesis. Adult C57BL/6 mice were fed with chow containing 5% (w/w) 4-MU for 6 months, at a dose ~6.7 mg/g/day. The oral administration of 4-MU reduced the glycosaminoglycan level in the brain to 72% and the spinal cord to 50% when compared to the controls. Spontaneous object recognition test (SOR) performed at 2, 3, 6 and 7 months showed a significant increase in SOR score in the 6-months treatment group 24 h after object presentation. The effect however did not persist in the washout group (1-month post treatment). Immunohistochemistry confirmed a reduction of PNNs, with shorter and less arborization of aggrecan staining around dendrites in hippocampus after 6 months of 4-MU treatment. Histopathological examination revealed mild atrophy in articular cartilage but it did not affect the motor performance as demonstrated in rotarod test. In conclusion, systemic oral administration of 4-MU for 6 months reduced PNN formation around neurons and enhanced memory retention in mice. However, the memory enhancement was not sustained despite the reduction of PNNs, possibly due to the lack of memory enhancement training during the washout period. Our results suggest that 4-MU treatment might offer a strategy for PNN modulation in memory enhancement.

Endoscopic Coregistered Ultrasound Imaging and Precision Histotripsy: Initial In Vivo Evaluation.

Objective. Initial performance evaluation of a system for simultaneous high-resolution ultrasound imaging and focused mechanical submillimeter histotripsy ablation in rat brains. Impact Statement. This study used a novel combination of high-resolution imaging and histotripsy in an endoscopic form. This would provide neurosurgeons with unprecedented accuracy in targeting and executing nonthermal ablations in minimally invasive surgeries. Introduction. Histotripsy is a safe and effective nonthermal focused ablation technique. However, neurosurgical applications, such as brain tumor ablation, are difficult due to the presence of the skull. Current devices are too large to use in the minimally invasive approaches surgeons prefer. We have developed a combined imaging and histotripsy endoscope to provide neurosurgeons with a new tool for this application. Methods. The histotripsy component had a 10 mm diameter, operating at 6.3 MHz. Affixed within a cutout hole in its center was a 30 MHz ultrasound imaging array. This coregistered pair was used to ablate brain tissue of anesthetized rats while imaging. Histological sections were examined, and qualitative descriptions of ablations and basic shape descriptive statistics were generated. Results. Complete ablations with submillimeter area were produced in seconds, including with a moving device. Ablation progress could be monitored in real time using power Doppler imaging, and B-mode was effective for monitoring post-ablation bleeding. Collateral damage was minimal, with a 100 µm maximum distance of cellular damage from the ablation margin. Conclusion. The results demonstrate a promising hardware suite to enable precision ablations in endoscopic procedures or fundamental preclinical research in histotripsy, neuroscience, and cancer.

Chondroitin 6-sulphate is required for neuroplasticity and memory in ageing.

Perineuronal nets (PNNs) are chondroitin sulphate proteoglycan-containing structures on the neuronal surface that have been implicated in the control of neuroplasticity and memory. Age-related reduction of chondroitin 6-sulphates (C6S) leads to PNNs becoming more inhibitory. Here, we investigated whether manipulation of the chondroitin sulphate (CS) composition of the PNNs could restore neuroplasticity and alleviate memory deficits in aged mice. We first confirmed that aged mice (20-months) showed memory and plasticity deficits. They were able to retain or regain their cognitive ability when CSs were digested or PNNs were attenuated. We then explored the role of C6S in memory and neuroplasticity. Transgenic deletion of chondroitin 6-sulfotransferase (chst3) led to a reduction of permissive C6S, simulating aged brains. These animals showed very early memory loss at 11 weeks old. Importantly, restoring C6S levels in aged animals rescued the memory deficits and restored cortical long-term potentiation, suggesting a strategy to improve age-related memory impairment.