First Organoid Intelligence (OI) workshop to form an OI community.

The brain is arguably the most powerful computation system known. It is extremely efficient in processing large amounts of information and can discern signals from noise, adapt, and filter faulty information all while running on only 20 watts of power. The human brain's processing efficiency, progressive learning, and plasticity are unmatched by any computer system. Recent advances in stem cell technology have elevated the field of cell culture to higher levels of complexity, such as the development of three-dimensional (3D) brain organoids that recapitulate human brain functionality better than traditional monolayer cell systems. Organoid Intelligence (OI) aims to harness the innate biological capabilities of brain organoids for biocomputing and synthetic intelligence by interfacing them with computer technology. With the latest strides in stem cell technology, bioengineering, and machine learning, we can explore the ability of brain organoids to compute, and store given information (input), execute a task (output), and study how this affects the structural and functional connections in the organoids themselves. Furthermore, understanding how learning generates and changes patterns of connectivity in organoids can shed light on the early stages of cognition in the human brain. Investigating and understanding these concepts is an enormous, multidisciplinary endeavor that necessitates the engagement of both the scientific community and the public. Thus, on Feb 22-24 of 2022, the Johns Hopkins University held the first Organoid Intelligence Workshop to form an OI Community and to lay out the groundwork for the establishment of OI as a new scientific discipline. The potential of OI to revolutionize computing, neurological research, and drug development was discussed, along with a vision and roadmap for its development over the coming decade.

Social status mediated variation in hypothalamic transcriptional profiles of male mice.

Animals of different social status exhibit variation in aggression, territorial and reproductive behavior as well as activity patterns, feeding, drinking and status signaling. This behavioral and physiological plasticity is coordinated by underlying changes in brain gene transcription. Using Tag-based RNA sequencing (Tag-seq), we explore RNA transcriptomes from the medial preoptic area (mPOA) and ventral hypothalamus (vHYP) of male mice of different social ranks in a dominance hierarchy and detect candidate genes and cellular pathways that underlie status-related plasticity. Within the mPOA, oxytocin (Oxt) and vasopressin (Avp) are more highly expressed in subdominant mice compared to other ranks, while nitric oxide synthase (Nos1) has lower expression in subdominant mice. Within the vHYP, we find that both orexigenic and anorexigenic genes involved in feeding behavior, including agouti-related peptide (Agrp), neuropeptide-Y (Npy), galanin (Gal), proopiomelanocortin (Pomc), and Cocaine- and Amphetamine-Regulated Transcript Protein prepropeptide (Cartpt), are less expressed in dominant animals compared to more subordinate ranks. We suggest that this may represent a reshaping of feeding circuits in dominant compared to subdominant and subordinate animals. Furthermore, we determine several genes that are positively and negatively associated with the level of despotism (aggression) in dominant males. Ultimately, we identify hypothalamic genes controlling feeding and social behaviors that are differentially transcribed across animals of varying social status. These changes in brain transcriptomics likely support phenotypic variation that enable animals to adapt to their current social status.

Behavioural and physiological plasticity in social hierarchies.

Individuals occupying dominant and subordinate positions in social hierarchies exhibit divergent behaviours, physiology and neural functioning. Dominant animals express higher levels of dominance behaviours such as aggression, territorial defence and mate-guarding. Dominants also signal their status via auditory, visual or chemical cues. Moreover, dominant animals typically increase reproductive behaviours and show enhanced spatial and social cognition as well as elevated arousal. These biobehavioural changes increase energetic demands that are met via shifting both energy intake and metabolism and are supported by coordinated changes in physiological systems including the hypothalamic-pituitary-adrenal and hypothalamic-pituitary-gonadal axes as well as altered gene expression and sensitivity of neural circuits that regulate these behaviours. Conversely, subordinate animals inhibit dominance and often reproductive behaviours and exhibit physiological changes adapted to socially stressful contexts. Phenotypic changes in both dominant and subordinate individuals may be beneficial in the short-term but lead to long-term challenges to health. Further, rapid changes in social ranks occur as dominant animals socially ascend or descend and are associated with dynamic modulations in the brain and periphery. In this paper, we provide a broad overview of how behavioural and phenotypic changes associated with social dominance and subordination are expressed in neural and physiological plasticity. This article is part of the theme issue 'The centennial of the pecking order: current state and future prospects for the study of dominance hierarchies'.

Effectiveness of the DROP training for military behavioral health providers targeting therapeutic alliance and premature dropout.

AbstractPremature discontinuation from behavioral health treatment is a major problem reducing effectiveness of care in military populations. A training was developed and delivered to 622 behavioral health providers across 15 sites within the Army behavioral healthcare system. The training taught two techniques to foster treatment engagement: (1) Progress Informed Treatment, consisting of reviewing symptom assessments and outcome measures, and (2) assessment and discussion of the treatment alliance via a paper survey given near the end of each session. Eighty-five percent of providers indicated the training was useful and 89% of providers incorporated a technique into their practice. Dropout before the fourth session was significantly reduced in the six months following training, from 72.5% to 67.1% in Service Members (SM; X2(1, N=9127) = 39.58, p < .001). In both the pre and post-training periods, providers working at the Master's level, SM aged 17 or 46 or older, and clients receiving a mood, PTSD, anxiety, adjustment, substance or childhood/adolescent psychiatric diagnosis experienced significantly less dropout, while SM aged 18-21 had significantly more dropout. This training is a feasible and available option to increase treatment engagement and improve treatment outcomes for service members.

Fabrication and Characterization of 3D Printed, 3D Microelectrode Arrays for Interfacing with a Peripheral Nerve-on-a-Chip.

We present a nontraditional fabrication technique for the realization of three-dimensional (3D) microelectrode arrays (MEAs) capable of interfacing with 3D cellular networks in vitro. The technology uses cost-effective makerspace microfabrication techniques to fabricate the 3D MEAs with 3D printed base structures with the metallization of the microtowers and conductive traces being performed by stencil mask evaporation techniques. A biocompatible lamination layer insulates the traces for realization of 3D microtower MEAs (250 µm base diameter, 400 µm height). The process has additionally been extended to realize smaller electrodes (30 µm × 30 µm) at a height of 400 µm atop the 3D microtower using laser micromachining of an additional silicon dioxide (SiO2) insulation layer. A 3D microengineered, nerve-on-a-chip in vitro model for recording and stimulating electrical activity of dorsal root ganglion (DRG) cells has further been integrated with the 3D MEA. We have characterized the 3D electrodes for electrical, chemical, electrochemical, biological, and chip hydration stability performance metrics. A decrease in impedance from 1.8 kΩ to 670 Ω for the microtower electrodes and 55 to 39 kΩ for the 30 µm × 30 µm microelectrodes can be observed for an electrophysiologically relevant frequency of 1 kHz upon platinum electroless plating. Biocompatibility assays on the components of the system resulted in a large range (∼3%-70% live cells), depending on the components. Fourier-transform infrared (FTIR) spectra of the resin material start to reveal possible compositional clues for the resin, and the hydration stability is demonstrated in in-vitro-like conditions for 30 days. The fabricated 3D MEAs are rapidly produced with minimal usage of a cleanroom and are fully functional for electrical interrogation of the 3D organ-on-a-chip models for high-throughput of pharmaceutical screening and toxicity testing of compounds in vitro.

Modeling chemotherapy-induced peripheral neuropathy using a Nerve-on-a-chip microphysiological system.

Organ-on-a-chip devices that mimic in vivo physiology have the potential to identify effects of chemical and drug exposure in early preclinical stages of drug development while relying less heavily on animal models. We have designed a hydrogel rat nerve-on-a-chip (RNoaC) construct that promotes axon growth analogous to mature nerve anatomy and is the first 3D in vitro model to collect electrophysiological and histomorphic metrics that are used to assess in vivo pathophysiology. Here we culture embryonic rat dorsal root ganglia (DRG) in the construct to demonstrate its potential as a preclinical assay for screening implications of nerve dysfunction in chemotherapy-induced peripheral neuropathy (CIPN). RNoaC constructs containing DRG explants from E15 rat pups were exposed to common chemotherapeutics: bortezomib, oxaliplatin, paclitaxel, or vincristine. After 7 days of treatment, axons were electrically stimulated to collect nerve conduction velocity (NCV) and the peak amplitude (AMP), which are two clinical electrophysiological metrics indicative of healthy or diseased populations. We observed decreased NCV and AMP in a dose-dependent manner across all drugs. At high drug concentrations, NCV and AMP were lower than control values by 10-60%. Histopathological analysis revealed that RNoaC exhibit hallmarks of peripheral neuropathy. IC50 values calculated from dose-response curves indicate significant decrease in function occurs before decrease in viability. Our data suggest electrophysiology recordings collected from our RNoaC platform can closely track subtle pathological changes in nerve function. The ability to collect clinically relevant data from RNoaCs suggests it can be an effective tool for in vitro preclinical screening of peripheral neuropathy.

Engineering a 3D functional human peripheral nerve in vitro using the Nerve-on-a-Chip platform.

Development of "organ-on-a-chip" systems for neuroscience applications are lagging due in part to the structural complexity of the nervous system and limited access of human neuronal & glial cells. In addition, rates for animal models in translating to human success are significantly lower for neurodegenerative diseases. Thus, a preclinical in vitro human cell-based model capable of providing critical clinical metrics such as nerve conduction velocity and histomorphometry are necessary to improve prediction and translation of in vitro data to successful clinical trials. To answer this challenge, we present an in vitro biomimetic model of all-human peripheral nerve tissue capable of showing robust neurite outgrowth (~5 mm), myelination of hNs by primary human Schwann cells (~5%), and evaluation of nerve conduction velocity (0.13-0.28 m/sec), previously unrealized for any human cell-based in vitro system. To the best of our knowledge, this Human Nerve-on-a-chip (HNoaC) system is the first biomimetic microphysiological system of myelinated human peripheral nerve which can be used for evaluating electrophysiological and histological metrics, the gold-standard assessment techniques previously only possible with in vivo studies.

Advancing nonclinical innovation and safety in pharmaceutical testing.

Nonclinical tests are considered crucial for understanding the safety of investigational medicines. However, the effective translation from nonclinical to human application is limited and must be improved. Drug development stakeholders are working to advance human-based in vitro and in silico methods that may be more predictive of human efficacy and safety in vivo because they enable scientists to model the direct interaction of drugs with human cells, tissues, and biological processes. Here, we recommend test-neutral regulations; increased funding for development and integration of human-based approaches; support for existing initiatives that advance human-based approaches; evaluation of new approaches using human data; establishment of guidelines for procuring human cells and tissues for research; and additional training and educational opportunities in human-based approaches.

Isolation of Human Adipose-Derived Stem Cells from Lipoaspirates.

Adipose tissue is as an abundant and accessible source of stem cells with multipotent properties suitable for tissue engineering and regenerative medical applications. Here, we describe methods from our own laboratory and the literature for the isolation and expansion of adipose-derived stem cells (ASCs). We present a large scale procedure suitable for processing >100 mL volumes of lipoaspirate tissue specimens by collagenase digestion and a related procedure suitable for processing adipose tissue aspirates without digestion.

Effect of Cryopreservation on Human Adipose Tissue and Isolated Stromal Vascular Fraction Cells: In Vitro and In Vivo Analyses.

BACKGROUND: Adipose tissue is a source of adipose-derived stromal/stem cells for tissue engineering and reconstruction and a tissue source for fat grafts. Although liposuction is a simple procedure for the harvest of adipose tissue, the repetition of this surgical intervention can cause adverse effects to the patient and can be a limiting factor for immediate use. Cryopreservation can avoid the morbidity associated with repetitive liposuction, allowing the use of stored tissue after the initial harvest procedure. This article focuses on the characterization of fresh and cryopreserved human adipose tissue. METHODS: Lipoaspirates from eight donors were processed as fresh adipose tissue or cryopreserved for 4 to 6 weeks. Fresh and cryopreserved tissues were collagenase digested and the stromal vascular fraction cells were characterized immediately or cryopreserved. Characterization was based on stromal vascular fraction cell proliferation and immunophenotype. In vivo fat grafting was performed in C57BL/6 green fluorescent protein mice to analyze morphology of the tissue and its adiposity using confocal microscopy, histochemical staining (i.e., hematoxylin and eosin and Masson trichrome), and immunohistochemistry (i.e., green fluorescent protein, perilipin, and CD31). RESULTS: Although tissue and stromal vascular fraction cell cryopreservation reduced the total cell yield, the remaining viable cells retained their adhesive and proliferative properties. The stromal vascular fraction cell immunophenotype showed a significant reduction in the hematopoietic surface markers and increased expression of stromal and adipogenic markers following cryopreservation. In vivo cryopreserved fat grafts showed morphology similar to that of freshly implanted fat grafts. CONCLUSION: In this study, the authors demonstrated that cryopreserved adipose tissue is a potential source of stromal vascular fraction cells and a suitable source for fat grafts.

3D Neural Culture in Dual Hydrogel Systems.

3D in vitro culture systems may yield physiological outcomes that more closely approximate in vivo behavior. A number of fabrication techniques and hydrogel scaffold materials are available to researchers, but often their implementation is complex and seemingly prohibitive. Herein, we describe a simplistic and adaptable dual hydrogel photolithography method utilized to engineer advanced in vitro systems for studies of neuronal development and characterization.

Isolated node engineering of neuronal systems using laser direct write.

Current limitations to the engineering of ex vivo and in vitro neural environments are hampering the ability to understand underlying neurophysiology. High levels of spatial specificity, reproducibility and viability have been previously reported using laser direct write (LDW) to print cells. However, despite the significant need no one has yet reported laser assisted printing of primary mammalian neuronal cells, an inherently sensitive but critically important population. Herein, we describe the use of LDW to reproducibly and accurately pattern viable dorsal root ganglion (DRG) neurons and supportive cells capable of neural outgrowth and network formation. Our demonstrated ability to engineer and control distinct micro-environmental components unlocks the potential for high throughput experiments to both understand underlying physiology and investigate therapeutic interventions.

Postnatal maternal care predicts divergent weaning strategies and the development of social behavior.

Maternal care experienced during postnatal development predicts long-term neurobiological and behavioral outcomes. However, the cascade of behavioral changes that emerge in response to maternal care has not been elucidated. In the current study, we examine naturally occurring variation in postnatal licking/grooming (LG) in C57BL/6J mice to determine its impact on preweaning maternal and pup behavior, the weaning process, the pace of developmental change, the emergence of social behavior, and indices of anxiety-like behavior in adulthood. Our analyses indicate that lower postnatal LG is associated with truncated and more infrequent maternal behavior during the preweaning period. Moreover, compared to High LG dams, Low LG dams are observed to actively wean their offspring sooner and have offspring that play more frequently. The heightened pace of developmental change observed in offspring of Low LG dams suggests a more rapid transition to behavioral and nutritional independence, which could have implications for future reproductive strategies.

Microengineered peripheral nerve-on-a-chip for preclinical physiological testing.

The use of advanced in vitro testing is a powerful tool to develop predictive cellular assays suitable for improving the high attrition rates of novel pharmaceutical compounds. A microscale, organotypic model of nerve tissue with physiological measures that mimic clinical nerve compound action potential (CAP) and nerve fiber density (NFD) tests may be more predictive of clinical outcomes, enabling a more cost-effective approach for selecting promising lead compounds with higher chances of late-stage success. However, the neurological architecture, physiology, and surrounding extracellular matrix are hard to mimic in vitro. Using a dual hydrogel construct and explants from rat embryonic dorsal root ganglia, the present study describes an in vitro method for electrophysiological recording of intra- and extra-cellular recordings using a spatially-controlled, microengineered sensory neural fiber tract. Specifically, these 3D neural cultures exhibit both structural and functional characteristics that closely mimic those of afferent sensory peripheral fibers found in vivo. Our dual hydrogel system spatially confines growth to geometries resembling nerve fiber tracts, allowing for a high density of parallel, fasciculated neural growth. Perhaps more importantly, outputs resembling clinically relevant test criteria, including the measurement of CAP and NFD are possible through our advanced model. Moreover, the 3D hydrogel constructs allow flexibility in incorporated cell type, geometric fabrication, and electrical manipulation, providing a viable assay for systematic culture, perturbation, and testing of biomimetic neural growth for mechanistic studies necessitating physiologically-relevant readouts.

The Relative Functionality of Freshly Isolated and Cryopreserved Human Adipose-Derived Stromal/Stem Cells.

The capability of multipotent mesenchymal stem cells to maintain cell viability, phenotype and differentiation ability upon thawing is critical if they are to be banked and used for future therapeutic purposes. In the present study, we examined the effect of 9-10 months of cryostorage on the morphology, immunophenotype, colony-forming unit (CFU) and differentiation capacity of fresh and cryopreserved human adipose-derived stromal/stem cells (ASCs) from the same donors. Cryopreservation did not reduce the CFU frequency and the expression levels of CD29, CD73, CD90 and CD105 remained unchanged with the exception of CD34 and CD45; however, the differentiation capacity of cryopreserved ASCs relative to fresh cells was significantly reduced. While our findings suggest that future studies are warranted to improve cryopreservation methods and agents, cryopreserved ASCs retain sufficient features to ensure their practical utility for both research and clinical applications.

ECG Case of the Month. Out-of-Hospital Cardiac Arrest. DIAGNOSIS: Atrial fibrillation with a rapid ventricular response (150 beats/minute) and right bundle branch block.

A muscular-appearing 50-year-old man was found down in his home by family members. Paramedics documented pulseless electrical activity and began cardiopulmonary resuscitation that included placement of an endotracheal tube. The resuscitation was continued in the hospital emergency department (ED), and after 20 minutes, an arterial pulse returned. An electrocardiogram (ECG) was obtained (Figure 1). Meanwhile, a past history established that the patient was a personal trainer who seemed fit and healthy until 10 days earlier, when he came to the ED because he had begun to lose his balance and fall frequently. Computed tomography (CT) at that time revealed lytic lesions in the fifth lumbar vertebra and extensive retroperitoneal lymphadenopathy involving the aortic, iliac, and obturator chains and the perirectal region. Arrangements had then been made for outpatient workup of a presumed malignancy.

Sensory axon guidance with semaphorin 6A and nerve growth factor in a biomimetic choice point model.

The direct effect of guidance cues on developing and regenerating axons in vivo is not fully understood, as the process involves a multiplicity of attractive and repulsive signals, presented both as soluble and membrane-bound ligands. A better understanding of axon guidance is critical to functional recovery following injury to the nervous system through improved outgrowth and mapping of damaged nerves. Due to their implications as inhibitors to central nervous system regeneration, we investigated the repulsive properties of semaphorin 6A and ephrin-B3 on E15 rat dorsal root ganglion explants, as well as possible interactions with soluble gradients of chemoattractive nerve growth factor (NGF). We employed a 3D biomimetic in vitro choice point model, which enabled the simple and rapid preparation of patterned gel growth matrices with quantifiable presentation of guidance cues in a specifiable manner that resembles the in vivo presentation of soluble and/or immobilized ligands. Neurites demonstrated an inhibitory response to immobilized Sema6A by lumbosacral dorsal root ganglion explants, while no such repulsion was observed for immobilized ephrin-B3 by explants at any spinal level. Interestingly, Sema6A inhibition could be partially attenuated in a concentration-dependent manner through the simultaneous presentation of soluble NGF gradients. The in vitro model described herein represents a versatile and valuable investigative tool in the quest for understanding developmental processes and improving regeneration following nervous system injury.

The evolution of genomic imprinting: theories, predictions and empirical tests.

The epigenetic phenomenon of genomic imprinting has motivated the development of numerous theories for its evolutionary origins and genomic distribution. In this review, we examine the three theories that have best withstood theoretical and empirical scrutiny. These are: Haig and colleagues' kinship theory; Day and Bonduriansky's sexual antagonism theory; and Wolf and Hager's maternal-offspring coadaptation theory. These theories have fundamentally different perspectives on the adaptive significance of imprinting. The kinship theory views imprinting as a mechanism to change gene dosage, with imprinting evolving because of the differential effect that gene dosage has on the fitness of matrilineal and patrilineal relatives. The sexual antagonism and maternal-offspring coadaptation theories view genomic imprinting as a mechanism to modify the resemblance of an individual to its two parents, with imprinting evolving to increase the probability of expressing the fitter of the two alleles at a locus. In an effort to stimulate further empirical work on the topic, we carefully detail the logic and assumptions of all three theories, clarify the specific predictions of each and suggest tests to discriminate between these alternative theories for why particular genes are imprinted.

Structural and molecular micropatterning of dual hydrogel constructs for neural growth models using photochemical strategies.

Chemotactic and haptotactic cues guide neurite growth toward appropriate targets by eliciting attractive or repulsive responses from the neurite growth cones. Here we present an integrated system allowing both structural and molecular micropatterning in dual hydrogel 3D tissue culture constructs for directing in vitro neuronal growth via structural, immobilized, and soluble guidance cues. These tissue culture constructs were fabricated into specifiable geometries using UV light reflected from a digital micromirror device acting as a dynamic photomask, resulting in dual hydrogel constructs consisting of a cell growth-restrictive polyethylene glycol (PEG) boundary with a cell growth-permissive interior of photolabile α-carboxy-2-nitrobenzyl cysteine agarose (CNBC-A). This CNBC-A was irradiated in discrete areas and subsequently tagged with maleimide-conjugated biomolecules. Fluorescent microscopy showed biomolecule binding only at the sites of irradiation in CNBC-A, and confocal microscopy confirmed 3D binding through the depth of the construct. Neurite outgrowth studies showed contained growth throughout CNBC-A. The diffusion rate of soluble fluorescein-bovine serum albumin through the dual hydrogel construct was controlled by PEG concentration and the distance between the protein source and the agarose interior; the timescale for a transient protein gradient changed with these parameters. These findings suggest the dual hydrogel system is a useful platform for manipulating a 3D in vitro microenvironment with patterned structural and molecular guidance cues for modeling neural growth and guidance.

Variation in maternal and anxiety-like behavior associated with discrete patterns of oxytocin and vasopressin 1a receptor density in the lateral septum.

The relationship between anxiety and maternal behavior has been explored across species using a variety of approaches, yet there is no clear consensus on the nature or direction of this relationship. In the current study, we have assessed stable individual differences in anxiety-like behavior in a large cohort (n=57) of female F2 hybrid mice. Using open-field behavior as a continuous and categorical (high vs. low) measure we examined the relationship between the anxiety-like behavior of virgin F2 females and the subsequent maternal behavior of these females. In addition, we quantified oxytocin (OTR) and vasopressin (V1a) receptor density within the lateral septum to determine the possible correlation with anxiety-like and maternal behavior. We find that, though activity levels within the open-field do predict latency to engage in pup retrieval, anxiety-like measures on this test are otherwise not associated with subsequent maternal behavior. OTR density in the dorsal lateral septum was found to be negatively correlated with activity levels in the open-field and positively correlated with frequency of nursing behavior. V1a receptor density was significantly correlated with postpartum licking/grooming of pups. Though we do not find support for the hypothesis that individual differences in trait anxiety predict variation in maternal behavior, we do find evidence for the role of OTR and V1a receptors in predicting maternal behavior in mice and suggest possible methodological issues (such as distinguishing between trait and state anxiety) that will be a critical consideration for subsequent studies of the anxiety-maternal behavior relationship. This article is part of a Special Issue entitled Oxytocin, Vasopressin, and Social Behavior.