Positional Cloning of a Type 2 Diabetes Quantitative Trait Locus; Tomosyn-2, a Negative Regulator of Insulin Secretion

We previously mapped a type 2 diabetes (T2D) locus on chromosome 16 (Chr 16) in an F2 intercross from the BTBR T (+) tf (BTBR) Lepob/ob and C57BL/6 (B6) Lepob/ob mouse strains. Introgression of BTBR Chr 16 into B6 mice resulted in a consomic mouse with reduced fasting plasma insulin and elevated glucose levels. We derived a panel of sub-congenic mice and narrowed the diabetes susceptibility locus to a 1.6 Mb region. Introgression of this 1.6 Mb fragment of the BTBR Chr 16 into lean B6 mice (B6.16BT36–38) replicated the phenotypes of the consomic mice. Pancreatic islets from the B6.16BT36–38 mice were defective in the second phase of the insulin secretion, suggesting that the 1.6 Mb region encodes a regulator of insulin secretion. Within this region, syntaxin-binding protein 5-like (Stxbp5l) or tomosyn-2 was the only gene with an expression difference and a non-synonymous coding single nucleotide polymorphism (SNP) between the B6 and BTBR alleles. Overexpression of the b-tomosyn-2 isoform in the pancreatic β-cell line, INS1 (832/13), resulted in an inhibition of insulin secretion in response to 3 mM 8-bromo cAMP at 7 mM glucose. In vitro binding experiments showed that tomosyn-2 binds recombinant syntaxin-1A and syntaxin-4, key proteins that are involved in insulin secretion via formation of the SNARE complex. The B6 form of tomosyn-2 is more susceptible to proteasomal degradation than the BTBR form, establishing a functional role for the coding SNP in tomosyn-2. We conclude that tomosyn-2 is the major gene responsible for the T2D Chr 16 quantitative trait locus (QTL) we mapped in our mouse cross. Our findings suggest that tomosyn-2 is a key negative regulator of insulin secretion

High-Throughput Single-Cell Manipulation in Brain Tissue

The complexity of neurons and neuronal circuits in brain tissue requires the genetic manipulation, labeling, and tracking of single cells. However, current methods for manipulating cells in brain tissue are limited to either bulk techniques, lacking single-cell accuracy, or manual methods that provide single-cell accuracy but at significantly lower throughputs and repeatability. Here, we demonstrate high-throughput, efficient, reliable, and combinatorial delivery of multiple genetic vectors and reagents into targeted cells within the same tissue sample with single-cell accuracy. Our system automatically loads nanoliter-scale volumes of reagents into a micropipette from multiwell plates, targets and transfects single cells in brain tissues using a robust electroporation technique, and finally preps the micropipette by automated cleaning for repeating the transfection cycle. We demonstrate multi-colored labeling of adjacent cells, both in organotypic and acute slices, and transfection of plasmids encoding different protein isoforms into neurons within the same brain tissue for analysis of their effects on linear dendritic spine density. Our platform could also be used to rapidly deliver, both ex vivo and in vivo, a variety of genetic vectors, including optogenetic and cell-type specific agents, as well as fast-acting reagents such as labeling dyes, calcium sensors, and voltage sensors to manipulate and track neuronal circuit activity at single-cell resolution

Following the genes: a framework for animal modeling of psychiatric disorders

The number of individual cases of psychiatric disorders that can be ascribed to identified, rare, single mutations is increasing with great rapidity. Such mutations can be recapitulated in mice to generate animal models with direct etiological validity. Defining the underlying pathogenic mechanisms will require an experimental and theoretical framework to make the links from mutation to altered behavior in an animal or psychopathology in a human. Here, we discuss key elements of such a framework, including cell type-based phenotyping, developmental trajectories, linking circuit properties at micro and macro scales and definition of neurobiological phenotypes that are directly translatable to humans

Oxidative stress-driven parvalbumin interneuron impairment as a common mechanism in models of schizophrenia.

Parvalbumin inhibitory interneurons (PVIs) are crucial for maintaining proper excitatory/inhibitory balance and high-frequency neuronal synchronization. Their activity supports critical developmental trajectories, sensory and cognitive processing, and social behavior. Despite heterogeneity in the etiology across schizophrenia and autism spectrum disorder, PVI circuits are altered in these psychiatric disorders. Identifying mechanism(s) underlying PVI deficits is essential to establish treatments targeting in particular cognition. On the basis of published and new data, we propose oxidative stress as a common pathological mechanism leading to PVI impairment in schizophrenia and some forms of autism. A series of animal models carrying genetic and/or environmental risks relevant to diverse etiological aspects of these disorders show PVI deficits to be all accompanied by oxidative stress in the anterior cingulate cortex. Specifically, oxidative stress is negatively correlated with the integrity of PVIs and the extracellular perineuronal net enwrapping these interneurons. Oxidative stress may result from dysregulation of systems typically affected in schizophrenia, including glutamatergic, dopaminergic, immune and antioxidant signaling. As convergent end point, redox dysregulation has successfully been targeted to protect PVIs with antioxidants/redox regulators across several animal models. This opens up new perspectives for the use of antioxidant treatments to be applied to at-risk individuals, in close temporal proximity to environmental impacts known to induce oxidative stress

Dynamic Effective Connectivity of Inter-Areal Brain Circuits

Anatomic connections between brain areas affect information flow between neuronal circuits and the synchronization of neuronal activity. However, such structural connectivity does not coincide with effective connectivity (or, more precisely, causal connectivity), related to the elusive question “Which areas cause the present activity of which others?”. Effective connectivity is directed and depends flexibly on contexts and tasks. Here we show that dynamic effective connectivity can emerge from transitions in the collective organization of coherent neural activity. Integrating simulation and semi-analytic approaches, we study mesoscale network motifs of interacting cortical areas, modeled as large random networks of spiking neurons or as simple rate units. Through a causal analysis of time-series of model neural activity, we show that different dynamical states generated by a same structural connectivity motif correspond to distinct effective connectivity motifs. Such effective motifs can display a dominant directionality, due to spontaneous symmetry breaking and effective entrainment between local brain rhythms, although all connections in the considered structural motifs are reciprocal. We show then that transitions between effective connectivity configurations (like, for instance, reversal in the direction of inter-areal interactions) can be triggered reliably by brief perturbation inputs, properly timed with respect to an ongoing local oscillation, without the need for plastic synaptic changes. Finally, we analyze how the information encoded in spiking patterns of a local neuronal population is propagated across a fixed structural connectivity motif, demonstrating that changes in the active effective connectivity regulate both the efficiency and the directionality of information transfer. Previous studies stressed the role played by coherent oscillations in establishing efficient communication between distant areas. Going beyond these early proposals, we advance here that dynamic interactions between brain rhythms provide as well the basis for the self-organized control of this “communication-through-coherence”, making thus possible a fast “on-demand” reconfiguration of global information routing modalities

Nanotools for Neuroscience and Brain Activity Mapping

Neuroscience is at a crossroads. Great effort is being invested into deciphering specific neural interactions and circuits. At the same time, there exist few general theories or principles that explain brain function. We attribute this disparity, in part, to limitations in current methodologies. Traditional neurophysiological approaches record the activities of one neuron or a few neurons at a time. Neurochemical approaches focus on single neurotransmitters. Yet, there is an increasing realization that neural circuits operate at emergent levels, where the interactions between hundreds or thousands of neurons, utilizing multiple chemical transmitters, generate functional states. Brains function at the nanoscale, so tools to study brains must ultimately operate at this scale, as well. Nanoscience and nanotechnology are poised to provide a rich toolkit of novel methods to explore brain function by enabling simultaneous measurement and manipulation of activity of thousands or even millions of neurons. We and others refer to this goal as the Brain Activity Mapping Project. In this Nano Focus, we discuss how recent developments in nanoscale analysis tools and in the design and synthesis of nanomaterials have generated optical, electrical, and chemical methods that can readily be adapted for use in neuroscience. These approaches represent exciting areas of technical development and research. Moreover, unique opportunities exist for nanoscientists, nanotechnologists, and other physical scientists and engineers to contribute to tackling the challenging problems involved in understanding the fundamentals of brain function

Targeting cells with single vectors using multiple-feature Boolean logic

Precisely defining the roles of specific cell types is an intriguing frontier in the study of intact biological systems and has stimulated the rapid development of genetically encoded tools for observation and control. However, targeting these tools with adequate specificity remains challenging: most cell types are best defined by the intersection of two or more features such as active promoter elements, location and connectivity. Here we have combined engineered introns with specific recombinases to achieve expression of genetically encoded tools that is conditional upon multiple cell-type features, using Boolean logical operations all governed by a single versatile vector. We used this approach to target intersectionally specified populations of inhibitory interneurons in mammalian hippocampus and neurons of the ventral tegmental area defined by both genetic and wiring properties. This flexible and modular approach may expand the application of genetically encoded interventional and observational tools for intact-systems biology

Patient-specific analyses of deep tissue loads post transtibial amputation in residual limbs of multiple prosthetic users

Active transtibial amputation (TTA) patients are at risk for developing pressure ulcers (PU) and deep tissue injury (DTI) while using their prosthesis. It is therefore important to obtain knowledge of the mechanical state in the internal soft tissues of the residuum, as well as knowledge of the mechanical state upon its surface. Our aim was to apply patient-specific MRI-based non-linear finite element (FE) models to quantify internal strains in TTA prosthetic users ( n=5) during load-bearing. By further employing a strain injury threshold for skeletal muscle, we identified patients susceptible to DTI. The geometrical characteristics of the residuum of the TTA participants varied substantially between patients, e.g. the residuum lengths were 7.6, 8.1, 9.2, 11.5 and 13.3 cm. We generally found that internal strains were higher in the bone proximity than in the muscle flap periphery. The highest strains, which in some patients exceeded 50% (engineering strain) for compressive, tensile and shear strains, were found in the shortest residual limbs, i.e. the 7.6 and 8.1 cm-long limbs. Correspondingly, the lowest strains were found in the 13.3 cm-long residuum, which had the bulkiest muscle flap. Yet, even in the case of a long residuum, about a third of the soft tissue volume at the distal tibial proximity area was occupied by large (>5%) internal compressive, tensile and shear strains. For both patients with shorter residual limbs, the internal principal compressive strains above 5% occupied almost the entire distal tibial proximity area. For a patient whose distal tibial end was flat (non-beveled), internal strains were more uniformly distributed, compared to the strain distributions in the other models, where focal elevated strains accumulated in the bone proximity. We found no muscle strains above the immediate injury threshold, indicating that all patients were not at immediate risk for DTI. Two patients whose residuum fat padding was minimal to none, were the only ones identified as theoretically prone to DTI at long (>3 h) continuous weight-bearing periods. We conclude that there is a wide variability in internal mechanical conditions between residual limbs across subjects, which necessitates patient-specific quantitative analyses of internal mechanical states in TTA patients, to assess the mechanical performance of the reconstructed limb and in particular, the individual risk for deep PU or DTI

Surgical and Morphological Factors that Affect Internal Mechanical Loads in Soft Tissues of the Transtibial Residuum

The residual limb of transtibial amputation (TTA) prosthetic users is threatened daily by pressure ulcers (PU) and deep tissue injury (DTI) caused mainly by sustained mechanical strains and stresses. Several risk factors dominate the extent of internal tissue loads in the residuum. In this study, we developed a set of three-dimensional finite element (FE) models that were variants of a patient-specific FE model, built from magnetic resonance imaging scans. The set of FE modes was utilized to assess the impact of the following risk factors on the strain/stress distribution in the muscle flap: (i) the tibial length, (ii) the tibial bevelment, (iii) a fibular osteophyte, (iv) the mechanical properties of the muscle, and (v) scarring in different locations and depths. A total of 12 nonlinear FE model configurations, representing variations in these factors, were built and solved. We present herein calculations of compression, tension and shear strains and stresses, von Mises stresses, and strain energy density averaged in critical locations in the muscle flap as well as volumes of concentration of elevated stresses in these areas. Our results overall show higher stresses accumulating in the bone proximity rather than in outlying soft tissues. The longer bone configurations spread the loads toward the external surfaces of the muscle flap. When shortening the truncated bones from 11.2 to 9.2 cm, the von Mises stresses at the distal edges of the bones were relieved considerably (by up to 80%), which indicates a predicted decreased risk for DTI. Decreasing the tibial bevelment mildly, from 52.3° to 37.7° caused propagation of internal stresses from the bone proximity toward the more superficial soft tissues of the residuum, thereby also theoretically reducing the risk for DTI. An osteophyte at the distal fibular end increased the strain and stress distributions directly under the fibula but had little effect (<1%) on stresses at other sites, e.g., under the tibia. Elevation of muscle stiffness (instantaneous shear modulus increase from 8.5 to 16.2 kPa), simulating variation between patients, and muscle flap contraction or spasm, showed the most substantial effect by an acute rise of the von Mises stresses at the bone proximity. The mean von Mises stresses at the bone proximity were approximately twofold higher in the contracted/spastic muscle when compared to the flaccid muscle. Locating a surgical scar in different sites and depths of the residuum had the least influence on the overall loading of the muscle flap (where stresses changed by <7%). Pending further validation by epidemiological PU and DTI risk factor studies, the conclusions of this study can be incorporated as guidelines for TTA surgeons, physical therapists, prosthetists, and the TTA patients themselves to minimize the onset of PU and DTI in this population. Additionally, the present analyses can be used to guide or focus epidemiological research of PU and DTI risk factors in the TTA population