Intrinsic Cellular Properties Research Articles

Knockout of Cyclophilin-D Provides Partial Amelioration of Intrinsic and Synaptic Properties Altered by Mild Traumatic Brain Injury.

Mitochondria are central to cell survival and Ca2+ homeostasis due to their intracellular buffering capabilities. Mitochondrial dysfunction resulting in mitochondrial permeability transition pore (mPTP) opening has been reported after mild traumatic brain injury (mTBI). Cyclosporine A provides protection against the mPTP opening through its interaction with cyclophilin-D (CypD). A recent study has found that the extent of axonal injury after mTBI was diminished in neocortex in cyclophilin-D knockout (CypDKO) mice. Here we tested whether this CypDKO could also provide protection from the increased intrinsic and synaptic neuronal excitability previously described after mTBI in a mild central fluid percussion injury mice model. CypDKO mice were crossed with mice expressing yellow fluorescent protein (YFP) in layer V pyramidal neurons in neocortex to create CypDKO/YFP-H mice. Whole cell patch clamp recordings from axotomized (AX) and intact (IN) YFP+ layer V pyramidal neurons were made 1 and 2 days after sham or mTBI in slices from CypDKO/YFP-H mice. Both excitatory post synaptic currents (EPSCs) recorded in voltage clamp and intrinsic cellular properties, including action potential (AP), afterhyperpolarization (AHP), and depolarizing after potential (DAP) characteristics recorded in current clamp were evaluated. There was no significant difference between sham and mTBI for either spontaneous or miniature EPSC frequency, suggesting that CypDKO ameliorates excitatory synaptic abnormalities. There was a partial amelioration of intrinsic properties altered by mTBI. Alleviated were the increased slope of the AP frequency vs. injected current plot, the increased AP, AHP and DAP amplitudes. Other properties that saw a reversal that became significant in the opposite direction include the current rheobase and AP overshoot. The AP threshold remained depolarized and the input resistance remained increased in mTBI compared to sham. Additional altered properties suggest that the CypDKO likely has a direct effect on membrane properties, rather than producing a selective reduction of the effects of mTBI. These results suggest that inhibiting CypD after TBI is an effective strategy to reduce synaptic hyperexcitation, making it a continued target for potential treatment of network abnormalities.

Using a Semi-Automated Strategy to Develop Multi-Compartment Models That Predict Biophysical Properties of Interneuron-Specific 3 (IS3) Cells in Hippocampus.

Determining how intrinsic cellular properties govern and modulate neuronal input–output processing is a critical endeavor for understanding microcircuit functions in the brain. However, lack of cellular specifics and nonlinear interactions prevent experiments alone from achieving this. Building and using cellular models is essential in these efforts. We focus on uncovering the intrinsic properties of mus musculus hippocampal type 3 interneuron-specific (IS3) cells, a cell type that makes GABAergic synapses onto specific interneuron types, but not pyramidal cells. While IS3 cell morphology and synaptic output have been examined, their voltage-gated ion channel profile and distribution remain unknown. We combined whole-cell patch-clamp recordings and two-photon dendritic calcium imaging to examine IS3 cell membrane and dendritic properties. Using these data as a target reference, we developed a semi-automated strategy to obtain multi-compartment models for a cell type with unknown intrinsic properties. Our approach is based on generating populations of models to capture determined features of the experimental data, each of which possesses unique combinations of channel types and conductance values. From these populations, we chose models that most closely resembled the experimental data. We used these models to examine the impact of specific ion channel combinations on spike generation. Our models predict that fast delayed rectifier currents should be present in soma and proximal dendrites, and this is confirmed using immunohistochemistry. Further, without A-type potassium currents in the dendrites, spike generation is facilitated at more distal synaptic input locations. Our models will help to determine the functional role of IS3 cells in hippocampal microcircuits.

Co-culture model consisting of human brain microvascular endothelial and peripheral blood mononuclear cells

BackgroundNumerous systems exist to model the blood–brain barrier (BBB) with the goal of understanding the regulation of passage into the central nervous system (CNS) and the potential impact of selected insults on BBB function. These models typically focus on the intrinsic cellular properties of the BBB, yet studies of peripheral cell migration are often excluded due to technical restraints. New methodThis method allows for the study of in vitro cellular transmigration following exposure to any treatment of interest through optimization of co-culture conditions for the human brain microvascular endothelial cells (BMEC) cell line, hCMEC/D3, and primary human peripheral blood mononuclear cells (PBMCs). ResultshCMEC/D3 cells form functionally confluent monolayers on collagen coated polytetrafluoroethylene (PTFE) transwell inserts, as assessed by microscopy and tracer molecule (FITC-dextran (FITC-D)) exclusion. Two components of complete hCMEC/D3 media, EBM-2 base-media and hydrocortisone (HC), were determined to be cytotoxic to PBMCs. By combining the remaining components of complete hCMEC/D3 media with complete PBMC media a resulting co-culture media was established for use in hCMEC/D3—PBMC co-culture functional assays. Comparison with existing methodsThrough this method, issues of extensive differences in culture media conditions are resolved allowing for treatments and functional assays to be conducted on the two cell populations co-cultured simultaneously. ConclusionDescribed here is an in vitro co-culture model of the BBB, consisting of the hCMEC/D3 cell line and primary human PBMCs. The co-culture media will now allow for the study of exposure to potential insults to BBB function over prolonged time courses.

Mesangial cells from patients with IgA nephropathy have increased susceptibility to galactose-deficient IgA1.

BackgroundIgA nephropathy (IgAN) is the most common glomerulonephritis in the world, affecting close to a million people. Circulating galactose-deficient IgA (gd-IgA), present in patients with IgAN, form immune complex deposits in the glomerular mesangium causing local proliferation and matrix expansion. Intriguing though, individuals having gd-IgA deposits in the kidneys do not necessarily have signs of glomerular disease. Recurrence of IgAN only occurs in less than half of transplanted patients with IgAN, indicating that gd-IgA is not the only factor driving the disease. We hypothesize that, in addition to IgA complexes, patients with IgAN possess a subtype of mesangial cells highly susceptible to gd-IgA induced cell proliferation.MethodsTo test the hypothesis, we designed a technique to culture primary mesangial cells from renal biopsies obtained from IgAN patients and controls. The cell response to gd-IgA treatment was then measured both on gene and protein level and the proliferation rate of the cells in response to PDGF was investigated.ResultsWhen treated with gd-IgA, mesangial cells from patients with IgAN express and release more PDGF compared to controls. In addition, the mesangial cells from patients with IgAN were more responsive to treatment with PDGF resulting in an increased proliferation rate of the cells compared to control. Mesangial cells cultured from patients with IgAN expressed and released more IL-6 than controls and had a higher expression of matrix genes. Both mesangial cells derived from patients with IgAN and controls increased their expressed TGFβ1 and CCL5 when treated with gd-IgA.ConclusionWe conclude that mesangial cells derived from IgAN patients have a mesangioproliferative phenotype with increased reactivity to IgA and that these cellular intrinsic properties may be important for the development of IgA nephropathy.

Mechanisms Underlying Adaptation of Respiratory Network Activity to Modulatory Stimuli in the Mouse Embryo.

Breathing is a rhythmic behavior that requires organized contractions of respiratory effector muscles. This behavior must adapt to constantly changing conditions in order to ensure homeostasis, proper body oxygenation, and CO2/pH regulation. Respiratory rhythmogenesis is controlled by neural networks located in the brainstem. One area considered to be essential for generating the inspiratory phase of the respiratory rhythm is the preBötzinger complex (preBötC). Rhythmogenesis emerges from this network through the interplay between the activation of intrinsic cellular properties (pacemaker properties) and intercellular synaptic connections. Respiratory activity continuously changes under the impact of numerous modulatory substances depending on organismal needs and environmental conditions. The preBötC network has been shown to become active during the last third of gestation. But only little is known regarding the modulation of inspiratory rhythmicity at embryonic stages and even less on a possible role of pacemaker neurons in this functional flexibility during the prenatal period. By combining electrophysiology and calcium imaging performed on embryonic brainstem slice preparations, we provide evidence showing that embryonic inspiratory pacemaker neurons are already intrinsically sensitive to neuromodulation and external conditions (i.e., temperature) affecting respiratory network activity, suggesting a potential role of pacemaker neurons in mediating rhythm adaptation to modulatory stimuli in the embryo.

H-Channels Affect Frequency, Power and Amplitude Fluctuations of Neuronal Network Oscillations.

Oscillations in network activity are ubiquitous in the brain and are involved in diverse cognitive functions. Oscillation characteristics, such as power, frequency, and temporal structure, depend on both network connectivity and intrinsic cellular properties, such as ion channel composition. An important class of channels, with key roles in regulating cell excitability, are h-channels. The h-current (Ih) is a slow, hyperpolarization-activated, depolarizing current that contributes to neuronal resonance and membrane potential. The impact of Ih on network oscillations, however, remains poorly understood. To elucidate the network effects of Ih, we used a computational model of a generic oscillatory neuronal network consisting of inhibitory and excitatory cells that were externally driven by excitatory action potentials and sustained depolarizing currents. We found that Ih increased the oscillation frequency and, in combination with external action potentials, representing input from areas outside the network, strongly decreased the synchrony of firing. As a consequence, the oscillation power and the duration of episodes during which the network exhibited high-amplitude oscillations were greatly reduced in the presence of Ih. Our results suggest that modulation of Ih or impaired expression of h-channels, as observed in epilepsy, could, by affecting oscillation dynamics, markedly alter network-level activity and potentially influence oscillation-dependent cognitive processes such as learning, memory and attention.

Constriction Channel Based Single-Cell Mechanical Property Characterization

This mini-review presents recent progresses in the development of microfluidic constriction channels enabling high-throughput mechanical property characterization of single cells. We first summarized the applications of the constriction channel design in quantifying mechanical properties of various types of cells including red blood cells, white blood cells, and tumor cells. Then we highlighted the efforts in modeling the cellular entry process into the constriction channel, enabling the translation of raw mechanical data (e.g., cellular entry time into the constriction channel) into intrinsic cellular mechanical properties such as cortical tension or Young’s modulus. In the end, current limitations and future research opportunities of the microfluidic constriction channels were discussed.

“Slow” skeletal muscles across vertebrate species

Skeletal muscle fibers are generally classified into two groups: slow (type I) and fast (type II). Fibers in each group are uniquely designed for specific locomotory needs based on their intrinsic cellular properties and the types of motor neurons that innervate them. In this review, we will focus on the current concept of slow muscle fibers which, unlike the originally proposed version based purely on amphibian muscles, varies widely depending on the animal model system studied. We will discuss recent findings from zebrafish neuromuscular junction synapses that may provide the framework for establishing a more unified view of slow muscles across mammalian and non-mammalian species.

A positive feedback at the cellular level promotes robustness and modulation at the circuit level.

This article highlights the role of a positive feedback gating mechanism at the cellular level in the robustness and modulation properties of rhythmic activities at the circuit level. The results are presented in the context of half-center oscillators, which are simple rhythmic circuits composed of two reciprocally connected inhibitory neuronal populations. Specifically, we focus on rhythms that rely on a particular excitability property, the postinhibitory rebound, an intrinsic cellular property that elicits transient membrane depolarization when released from hyperpolarization. Two distinct ionic currents can evoke this transient depolarization: a hyperpolarization-activated cation current and a low-threshold T-type calcium current. The presence of a slow activation is specific to the T-type calcium current and provides a slow positive feedback at the cellular level that is absent in the cation current. We show that this slow positive feedback is required to endow the network rhythm with physiological modulation and robustness properties. This study thereby identifies an essential cellular property to be retained at the network level in modeling network robustness and modulation.

Transcutaneous induction of stimulus-timing-dependent plasticity in dorsal cochlear nucleus.

The cochlear nucleus (CN) is the first site of multisensory integration in the ascending auditory pathway. The principal output neurons of the dorsal cochlear nucleus (DCN), fusiform cells, receive somatosensory information relayed by the CN granule cells from the trigeminal and dorsal column pathways. Integration of somatosensory and auditory inputs results in long-term enhancement or suppression in a stimulus-timing-dependent manner. Here, we demonstrate that stimulus-timing-dependent plasticity (STDP) can be induced in DCN fusiform cells using paired auditory and transcutaneous electrical stimulation of the face and neck to activate trigeminal and dorsal column pathways to the CN, respectively. Long-lasting changes in fusiform cell firing rates persisted for up to 2 h after this bimodal stimulation, and followed Hebbian or anti-Hebbian rules, depending on tone duration, but not somatosensory stimulation location: 50 ms paired tones evoked predominantly Hebbian, while 10 ms paired tones evoked predominantly anti-Hebbian plasticity. The tone-duration-dependent STDP was strongly correlated with first inter-spike intervals, implicating intrinsic cellular properties as determinants of STDP. This study demonstrates that transcutaneous stimulation with precise auditory–somatosensory timing parameters can non-invasively induce fusiform cell long-term modulation, which could be harnessed in the future to moderate tinnitus-related hyperactivity in DCN.

T Cells Integrate Local and Global Cues to Discriminate between Structurally Similar Antigens.

T lymphocytes’ ability to discriminate between structurally related antigens has been attributed to the unique signaling properties of the T cell receptor. However, recent studies have suggested that the output of this discrimination process is conditioned by environmental cues. Here, we demonstrate how the IL-2 cytokine, collectively generated by strongly activated T cell clones, can induce weaker T cell clones to proliferate. We identify the PI3K pathway as being critical for integrating the antigen and cytokine responses and for controlling cell-cycle entry. We build a hybrid stochastic/deterministic computational model that accounts for such signal synergism and demonstrates quantitatively how T cells tune their cell-cycle entry according to environmental cytokine cues. Our findings indicate that antigen discrimination by T cells is not solely an intrinsic cellular property but rather a product of integration of multiple cues, including local cues such as antigen quality and quantity, to global ones like the extracellular concentration of inflammatory cytokines.

Investigating cell mechanics with atomic force microscopy.

Transmission of mechanical force is crucial for normal cell development and functioning. However, the process of mechanotransduction cannot be studied in isolation from cell mechanics. Thus, in order to understand how cells 'feel', we must first understand how they deform and recover from physical perturbations. Owing to its versatility, atomic force microscopy (AFM) has become a popular tool to study intrinsic cellular mechanical properties. Used to directly manipulate and examine whole and subcellular reactions, AFM allows for top-down and reconstitutive approaches to mechanical characterization. These studies show that the responses of cells and their components are complex, and largely depend on the magnitude and time scale of loading. In this review, we generally describe the mechanotransductive process through discussion of well-known mechanosensors. We then focus on discussion of recent examples where AFM is used to specifically probe the elastic and inelastic responses of single cells undergoing deformation. We present a brief overview of classical and current models often used to characterize observed cellular phenomena in response to force. Both simple mechanistic models and complex nonlinear models have been used to describe the observed cellular behaviours, however a unifying description of cell mechanics has not yet been resolved.

Does Cell Shape Determine Cell Fate?

Different types of cells, i.e. from different tissues, typically look quite different from each other. Even when cultured on two-dimensional surfaces like glass slides or tissue culture polystyrene under identical conditions, cells adopt different shapes. These shapes are in general functions of the cytoskeletal properties of those cells, itself a subset of what we call the “state” of the cell. Moreover the changes in cell shape upon perturbation of the surface or of the cells themselves should reflect their intrinsic cellular properties, i.e. the cell state. Significant evidence has accumulated that changes in shape can also alter cellular properties, at least for some cells. Our experiments suggest that for Mesenchymal Stem Cells (MSCs), shape perturbations have consequences for their differentiation into osteoblasts. Thus shape seemed linked to fate. These statements beg the question: is it possible to use cell shape to assess cell state? For example can we back-calculate the cytoskeletal properties of the cell from the way it looks on surfaces? This question becomes all the more interesting for cancer cells since cancer cells are known to have altered mechanical properties compared to normal cells, and invasive cancer cells appear to have altered mechanical properties compared to non-invasive cancer cells. In this work we present a combination of experiments and statistical data analysis to try to begin to understand how cell shapes are affected by changes in surface properties or by perturbations of the cytoskeleton. We use fluorescent imaging to obtain the two-dimensional profile of cells and novel Third Harmonic Generation methods to obtain three-dimensional images on cells on substrates. We use these experiments to infer how the cell shape of cancer cells could be associated with their invasive properties. We discuss some rudimentary mathematical models based on these results.

The Morphogenetic Role of Apoptosis.

Beyond safeguarding the organism from cell misbehavior and controlling cell number, apoptosis (or programmed cell death) plays key roles during animal development. In particular, it has long been acknowledged that apoptosis participates in tissue remodeling. Yet, until recently, this contribution to morphogenesis was considered as "passive," consisting simply in the local removal of unnecessary cells leading to a new shape. In recent years, applying live imaging methods to study the dynamics of apoptosis in various contexts has considerably modified our vision, revealing that in fact, dying cells remodel their neighborhood actively. Here, we first focus on the intrinsic cellular properties of apoptotic cells during their dismantling, in particular the role of the cytoskeleton during their characteristic morphological changes. Second, we review the various roles of apoptosis during developmental morphogenetic processes and pinpoint the crucial role of live imaging in revealing new concepts, in particular apoptosis as a generator of mechanical forces to control tissue dynamics.

Spatiotemporal control of gene expression using microfluidics

Accurate spatiotemporal regulation of genetic expression and cell microenvironment are both essential to epithelial morphogenesis during development, wound healing and cancer. In vivo, this is achieved through the interplay between intrinsic cellular properties and extrinsic signals. Amongst these, morphogen gradients induce specific concentration- and time-dependent gene expression changes that influence a target cell's fate. As systems biology attempts to understand the complex mechanisms underlying morphogenesis, the lack of experimental setup to recapitulate morphogen-induced patterning in vitro has become limiting. For this reason, we developed a versatile microfluidic-based platform to control the spatiotemporal delivery of chemical gradients to tissues grown in Petri dishes. Using this setup combined with a synthetic inducible gene expression system, we were able to restrict a target gene's expression within a confluent epithelium to bands of cells as narrow as four cell diameters with a one cell diameter accuracy. Applied to the targeted delivery of growth factor gradients to a confluent epithelium, this method further enabled the localized induction of epithelial-mesenchymal transitions and associated morphogenetic changes. Our approach paves the way for replicating in vitro the morphogen gradients observed in vivo to determine the relative contributions of known intrinsic and extrinsic factors in differential tissue patterning, during development and cancer. It could also be readily used to spatiotemporally control cell differentiation in ES/iPS cell cultures for re-engineering of complex tissues. Finally, the reversibility of the microfluidic chip assembly allows for pre- and post-treatment sample manipulations and extends the range of patternable samples to animal explants.

Mechanisms and Functions of Theta Rhythms

The theta rhythm is one of the largest and most sinusoidal activity patterns in the brain. Here I survey progress in the field of theta rhythms research. I present arguments supporting the hypothesis that theta rhythms emerge owing to intrinsic cellular properties yet can be entrained by several theta oscillators throughout the brain. I review behavioral correlates of theta rhythms and consider how these correlates inform our understanding of theta rhythms' functions. I discuss recent work suggesting that one function of theta is to package related information within individual theta cycles for more efficient spatial memory processing. Studies examining the role of theta phase precession in spatial memory, particularly sequence retrieval, are also summarized. Additionally, I discuss how interregional coupling of theta rhythms facilitates communication across brain regions. Finally, I conclude by summarizing how theta rhythms may support cognitive operations in the brain, including learning.

Development of on-off spiking in superior paraolivary nucleus neurons of the mouse

The superior paraolivary nucleus (SPON) is a prominent cell group in the auditory brain stem that has been increasingly implicated in representing temporal sound structure. Although SPON neurons selectively respond to acoustic signals important for sound periodicity, the underlying physiological specializations enabling these responses are poorly understood. We used in vitro and in vivo recordings to investigate how SPON neurons develop intrinsic cellular properties that make them well suited for encoding temporal sound features. In addition to their hallmark rebound spiking at the stimulus offset, SPON neurons were characterized by spiking patterns termed onset, adapting, and burst in response to depolarizing stimuli in vitro. Cells with burst spiking had some morphological differences compared with other SPON neurons and were localized to the dorsolateral region of the nucleus. Both membrane and spiking properties underwent strong developmental regulation, becoming more temporally precise with age for both onset and offset spiking. Single-unit recordings obtained in young mice demonstrated that SPON neurons respond with temporally precise onset spiking upon tone stimulation in vivo, in addition to the typical offset spiking. Taken together, the results of the present study demonstrate that SPON neurons develop sharp on-off spiking, which may confer sensitivity to sound amplitude modulations or abrupt sound transients. These findings are consistent with the proposed involvement of the SPON in the processing of temporal sound structure, relevant for encoding communication cues.

Label-Free Enrichment of Adrenal Cortical Progenitor Cells Using Inertial Microfluidics

Passive and label-free isolation of viable target cells based on intrinsic biophysical cellular properties would allow for cost savings in applications where molecular biomarkers are known as well as potentially enable the separation of cells with little-to-no known molecular biomarkers. We have demonstrated the purification of adrenal cortical progenitor cells from digestions of murine adrenal glands utilizing hydrodynamic inertial lift forces that single cells and multicellular clusters differentially experience as they flow through a microchannel. Fluorescence staining, along with gene expression measurements, confirmed that populations of cells collected in different outlets were distinct from one another. Furthermore, primary murine cells processed through the device remained highly viable and could be cultured for 10 days in vitro. The proposed target cell isolation technique can provide a practical means to collect significant quantities of viable intact cells required to translate stem cell biology to regenerative medicine in a simple label-free manner.

Excitability of jcBNST Neurons Is Reduced in Alcohol-Dependent Animals during Protracted Alcohol Withdrawal

Alcohol dependence and withdrawal has been shown to cause neuroadaptive changes at multiple levels of the nervous system. At the neuron level, adaptations of synaptic connections have been extensively studied in a number of brain areas and accumulating evidence also shows the importance of alcohol dependence-related changes in the intrinsic cellular properties of neurons. At the same time, it is still largely unknown how such neural adaptations impact the firing and integrative properties of neurons. To address these problems, here, we analyze physiological properties of neurons in the bed nucleus of stria terminalis (jcBNST) in animals with a history of alcohol dependence. As a comprehensive approach, first we measure passive and active membrane properties of neurons using conventional current clamp protocols and then analyze their firing responses under the action of simulated synaptic bombardment via dynamic clamp. We find that most physiological properties as measured by DC current injection are barely affected during protracted withdrawal. However, neuronal excitability as measured from firing responses under simulated synaptic inputs with the dynamic clamp is markedly reduced in all 3 types of jcBNST neurons. These results support the importance of studying the effects of alcohol and drugs of abuse on the firing properties of neurons with dynamic clamp protocols designed to bring the neurons into a high conductance state. Since the jcBNST integrates excitatory inputs from the basolateral amygdala (BLA) and cortical inputs from the infralimbic and the insular cortices and in turn is believed to contribute to the inhibitory input to the central nucleus of the amygdala (CeA) the reduced excitability of the jcBNST during protracted withdrawal in alcohol-dependent animals will likely affect ability of the jcBNST to shape the activity and output of the CeA.

Phase-locking, quasiperiodicity and chaos in periodically driven noisy neuronal models: a spectral approach

In auditory experiments neurons are often driven by periodic or periodically modulated inputs. The response of the neurons is often periodic and phase-locked to the stimulus. However, this is not always the case. It is well-known that even simple deterministic models can exhibit quasiperiodic behavior, with no clear relationship between the phases of the stimulus and response. Moreover, the biological situation is further complicated by presence of noise in the periodic inputs and intrinsic cellular properties, e.g. firing rate adaptation. We develop analytical tools for detection of parameter regimes corresponding to different locking behaviors in more detailed stochastic biophysical models, so that these regimes can be avoided or chosen as necessary. In an effort to develop a mathematical theory applicable to the above biologically-motivated project on phase-locking and related phenomena, we have developed a spectral approach to stochastic circle maps. A stochastic circle map is defined as a Markov chain on the circle. This abstract class of objects includes a wide range of models for firing times of periodically forced noisy neuronal models. We define and analyze some dynamic properties of the system such as stochastic phase locking and stochastic bifurcation cascades. The main tool is analysis of the spectrum and eigenspaces of the transition operator of the Markov chain. We relate the dynamics of the stochastic system to those of a few identifiable finite state deterministic dynamical systems, the dynamics of which can in turn be deduced from the spectral properties of the corresponding matrices. We also show that the firing regime of the stochastic system can be determined by the shape of its eigenvalue “cloud.”