Signaling to the nucleus by an L-type calcium channel-calmodulin complex through the MAP kinase pathway.

Increases in the intracellular concentration of calcium ([Ca2+]i) activate various signaling pathways that lead to the expression of genes that are essential for dendritic development, neuronal survival, and synaptic plasticity. The mode of Ca2+ entry into a neuron plays a key role in determining which signaling pathways are activated and thus specifies the cellular response to Ca2+. Ca2+ influx through L-type voltage-activated channels (LTCs) is particularly effective at activating transcription factors such as CREB and MEF-2. We developed a functional knock-in technique to investigate the features of LTCs that specifically couple them to the signaling pathways that regulate gene expression. We found that an isoleucine-glutamine ("IQ") motif in the carboxyl terminus of the LTC that binds Ca2+-calmodulin (CaM) is critical for conveying the Ca2+ signal to the nucleus. Ca2+-CaM binding to the LTC was necessary for activation of the Ras/mitogen-activated protein kinase (MAPK) pathway, which conveys local Ca2+ signals from the mouth of the LTC to the nucleus. CaM functions as a local Ca2+ sensor at the mouth of the LTC that activates the MAPK pathway and leads to the stimulation of genes that are essential for neuronal survival and plasticity.

Calcium regulation of neuronal gene expression.

Plasticity is a remarkable feature of the brain, allowing neuronal structure and function to accommodate to patterns of electrical activity. One component of these long-term changes is the activity-driven induction of new gene expression, which is required for both the long-lasting long-term potentiation of synaptic transmission associated with learning and memory, and the activity dependent survival events that help to shape and wire the brain during development. We have characterized molecular mechanisms by which neuronal membrane depolarization and subsequent calcium influx into the cytoplasm lead to the induction of new gene transcription. We have identified three points within this cascade of events where the specificity of genes induced by different types of stimuli can be regulated. By using the induction of the gene that encodes brain-derived neurotrophic factor (BDNF) as a model, we have found that the ability of a calcium influx to induce transcription of this gene is regulated by the route of calcium entry into the cell, by the pattern of phosphorylation induced on the transcription factor cAMP-response element (CRE) binding protein (CREB), and by the complement of active transcription factors recruited to the BDNF promoter. These results refine and expand the working model of activity-induced gene induction in the brain, and help to explain how different types of neuronal stimuli can activate distinct transcriptional responses.

Gene regulation mediated by calcium signals in T lymphocytes.

Modulation of many signaling pathways in antigen-stimulated T and B cells results in global changes in gene expression. Here we investigate the contribution of calcium signaling to gene expression in T cells using cell lines from two severe-combined immunodeficiency patients with several cytokine deficiencies and diminished activation of the transcription factor NFAT nuclear factor of activated T cells. These T cells show a strong defect in transmembrane calcium influx that is also apparent in their B cells and fibroblasts. DNA microarray analysis of calcium entry-deficient and control T cells shows that Ca2+ signals both activate and repress gene expression and are largely transduced through the phosphatase calcineurin. We demonstrate an elaborate network of signaling pathways downstream of the T cell receptor, explaining the complexity of changes in gene expression during T cell activation.

Quantitative and qualitative control of antigen receptor signalling in tolerant B lymphocytes.

Lymphocyte antigen receptors, such as the B cell antigen receptor (BCR), have the ability to promote or inhibit immune responses. This functional plasticity is exemplified by BCR-induced mitosis in naïve but not tolerant B cells and is correlated with biochemical differences in the signals triggered by foreign and self antigens. Acute stimulation of naïve B cells with foreign antigen induces a biphasic Ca2+ flux, and activates nuclear signalling through NF-AT, NF-kappa B, JNK and ERK. In tolerant B lymphocytes, by contrast, self antigen triggers only a low Ca2+ plateau, NF-AT and ERK. After removal from self antigen, the BCRs on tolerant B cells reacquire the ability to stimulate a biphasic Ca2+ flux and to promote proliferation. The differences in nuclear signalling between naïve and tolerant cells is brought about in part by differences in the magnitude of the Ca2+ signal. A low, sustained Ca2+ signal, such as that seen in tolerant B cells, activates NF-AT, whereas, a high but transient Ca2+ spike, which resembles that triggered in naïve B cells, activates NF-kappa B and JNK. These findings demonstrate that the quantitative differences in Ca2+ signalling between naïve and tolerant B cells are reversible and contribute to the differential triggering of nuclear signals. The activation of selected transcription factors may in turn account for the different functional responses triggered in naïve and tolerant lymphocytes.

A fluorometric method for estimating the calcium content of internal stores.

The concentration of Ca2+ in intracellular stores is an important factor in many aspects of Ca2+ signaling, including the generation of Ca2+ spikes, oscillations and waves, control of mitochondrial respiration, and activation of store-operated Ca2+ channels. Here we describe a consistent method for estimating the content of stores, based on the release of stored Ca2+ by thapsigargin (TG) or ionomycin (IO). Once released from stores, Ca2+ elevates [Ca2+]i transiently before it is pumped across the plasma membrane. If the dependence of the pump rate on [Ca2+]i is known, then the kinetics and amplitude of the Ca2+ transient allows the total amount of releasable Ca2+ to be estimated. We develop this quantitative approach and validate its use in human T cells, in which the Ca2+ clearance rate is an approximately linear function of [Ca2+]i. Our results support the assumption that the ER Ca2+ leak in resting T cells is unregulated, i.e. its rate is proportional to luminal [Ca2+]. The characteristic time constant for basal Ca2+ release is 110-140 s, comparable to that for activation of Ca2+ release-activated Ca2+ (CRAC) channels by TG and consistent with the dependence of ICRAC on store depletion. This method for estimating store content may be useful for quantifying the overlap between functionally distinct stores and for defining the relation between store content and cellular responses.

Calcium oscillations increase the efficiency and specificity of gene expression.

Cytosolic calcium ([Ca2+]i) oscillations are a nearly universal mode of signalling in excitable and non-excitable cells. Although Ca2+ is known to mediate a diverse array of cell functions, it is not known whether oscillations contribute to the efficiency or specificity of signalling or are merely an inevitable consequence of the feedback control of [Ca2+]i. We have developed a Ca2+ clamp technique to investigate the roles of oscillation amplitude and frequency in regulating gene expression driven by the proinflammatory transcription factors NF-AT, Oct/OAP and NF-kappaB. Here we report that oscillations reduce the effective Ca2+ threshold for activating transcription factors, thereby increasing signal detection at low levels of stimulation. In addition, specificity is encoded by the oscillation frequency: rapid oscillations stimulate all three transcription factors, whereas infrequent oscillations activate only NF-kappaB. The genes encoding the cytokines interleukin (IL)-2 and IL-8 are also frequency-sensitive in a way that reflects their degree of dependence on NF-AT versus NF-kappaB. Our results provide direct evidence that [Ca2+]i oscillations increase both the efficacy and the information content of Ca2+ signals that lead to gene expression and cell differentiation.

Differential activation of transcription factors induced by Ca2+ response amplitude and duration.

An increase in the intracellular calcium ion concentration ([Ca2+]i) controls a diverse range of cell functions, including adhesion, motility, gene expression and proliferation. Calcium signalling patterns can occur as single transients, repetitive oscillations or sustained plateaux, but it is not known whether these patterns are responsible for encoding the specificity of cellular responses. We report here that the amplitude and duration of calcium signals in B lymphocytes controls differential activation of the pro-inflammatory transcriptional regulators NF-kappaB, c-Jun N-terminal kinase (JNK) and NFAT. NF-kappaB and JNK are selectively activated by a large transient [Ca2+]i rise, whereas NFAT is activated by a low, sustained Ca2+ plateau. Differential activation results from differences in the Ca2+ sensitivities and kinetic behaviour of the three pathways. Our results show how downstream effectors can decode information contained in the amplitude and duration of Ca2+ signals, revealing a mechanism by which a multifunctional second messenger such as Ca2+ can achieve specificity in signalling to the nucleus.

Different nuclear signals are activated by the B cell receptor during positive versus negative signaling.

It is not known how immunogenic versus tolerogenic cellular responses are signaled by receptors such as the B cell antigen receptor (BCR). Here we compare BCR signaling in naive cells that respond positively to foreign antigen and self-tolerant cells that respond negatively to self-antigen. In naive cells, foreign antigen triggered a large biphasic calcium response and activated nuclear signals through NF-AT, NF-kappa B, JNK, and ERK/pp90rsk. In tolerant B cells, self-antigen stimulated low calcium oscillations and activated NF-AT and ERK/pp90rsk but not NF-kappa B or JNK. Self-reactive B cells lacking the phosphatase CD45 did not exhibit calcium oscillations or ERK/pp90rsk activation, nor did they repond negatively to self-antigen. These data reveal striking biochemical differences in BCR signaling to the nucleus during positive selection by foreign antigens and negative selection by self-antigens.

Signaling between intracellular Ca2+ stores and depletion-activated Ca2+ channels generates [Ca2+]i oscillations in T lymphocytes.

Stimulation through the antigen receptor (TCR) of T lymphocytes triggers cytosolic calcium ([Ca2+]i) oscillations that are critically dependent on Ca2+ entry across the plasma membrane. We have investigated the roles of Ca2+ influx and depletion of intracellular Ca2+ stores in the oscillation mechanism, using single-cell Ca2+ imaging techniques and agents that deplete the stores. Thapsigargin (TG; 5-25 nM), cyclopiazonic acid (CPA; 5-20 microM), and tert-butylhydroquinone (tBHQ; 80-200 microM), inhibitors of endoplasmic reticulum Ca(2+)-ATPases, as well as the Ca2+ ionophore ionomycin (5-40 nM), elicit [Ca2+]i oscillations in human T cells. The oscillation frequency is approximately 5 mHz (for ATPase inhibitors) to approximately 10 mHz (for ionomycin) at 22-24 degrees C. The [Ca2+]i oscillations resemble those evoked by TCR ligation in terms of their shape, amplitude, and an absolute dependence on Ca2+ influx. Ca(2+)-ATPase inhibitors and ionomycin induce oscillations only within a narrow range of drug concentrations that are expected to cause partial depletion of intracellular stores. Ca(2+)-induced Ca2+ release does not appear to be significantly involved, as rapid removal of extracellular Ca2+ elicits the same rate of [Ca2+]i decline during the rising and falling phases of the oscillation cycle. Both transmembrane Ca2+ influx and the content of ionomycin-releasable Ca2+ pools fluctuate in oscillating cells. From these data, we propose a model in which [Ca2+]i oscillations in T cells result from the interaction between intracellular Ca2+ stores and depletion-activated Ca2+ channels in the plasma membrane.

Identification of random responders on MMPI protocols.

In clinical practice, indicators of random or irrelevant responses on MMPI protocols are typically not examined, and, if obtained, assumed to be the result of either gross psychopathology or attempts to malinger. A sample of 40 computer-generated random profiles and 40 profiles of forensic outpatients were compared on validity scales, clinical scales, and scales specially designed to detect randomness (TR index and Carelessness Scale). Results of discriminant analysis indicated differentiating patterns of responses with random profiles generally elevated. Further, the accuracy of several clinical decisions rules were evaluated. The "F greater than 80 and TR greater than 4" rule was found to have the greatest clinical utility at correctly classifying random responders.

Scientific inquiry in forensic psychiatry.

Forensic psychiatry and psychology, with an extensive history of clinical practice, is only recently emerging as a new scientific specialty. It is confronted with both general scientific-methodological issues, as well as unique, empirically-based psycholegal applications. Aimed towards a broader, more flexible paradigm for understanding the basis of our scientific inquiry, the paper examines various research and methodological issues. As a model for connecting and understanding these complicated and interrelated issues, Holton's explicit and useful model of scientific structure is provided. The methodology, logical and statistical analysis, and themata are explored with regards to their potential impact on scientific inquiry in forensic psychiatry. This paper argues for several considerations in furthering research. From a methodological basis, the prospective researcher has to consider (1) whether the methodology is, in part, predetermining the results; (2) whether the results are a prediction of the inclusion criteria rather than an independent finding; (3) what balance between specificity and generalizability is designed within the research study; and (4) what is the impact of various methodological artifacts on the results and conclusions of forensic psychiatric research (e.g., demand characteristics, evaluation apprehension, experimenter bias, systems influence). Further, the forensic psychiatric researcher is asked to consider the basis of the "validity" of his research findings in comparison with "objective reality" from the perspective of logical and statistical analysis. Scientists within forensic psychiatry are asked to consider (1) whether to employ "best fit" or complementarity in understanding their results; (2) how to make explicit the steps in data transformation and redefinition within their study; (3) what are the implications of exclusive use of null hypothesis testing in establishing research results; and (4) what is the comparative utility of non-parametric and multivariate statistical procedures in studying and understanding experimental variables. Finally, in acknowledging the non-linear and sometimes self-justifying aspect of science, researchers are invited to examine their basic assumptions, and the self-perpetuating and constraining nature of unacknowledged themata, as well as their impact on forensic psychiatry. This paper is conceptualized as a movement towards articulating both general methodological issues and their unique application to forensic psychiatry. The brief exposition of Holton's model and review of illustrative research in forensic psychiatry constitutes one attempt to strengthen the scientific rigor of forensic psychiatric research.

An empirical approach to insanity evaluations.

Described the development of the Rogers Criminal Responsibility Assessment Scales (RCRAS) as an empirically based testing procedure specifically designed for use in insanity evaluations. A pretest of the RCRAS that employed 10 case vignettes and a preliminary study of 25 patients are reported. Initial results indicated that the RCRAS has satisfactory interrater reliability and successfully discriminated between those patients evaluated as sane and insane. Results of the discriminant analysis, MANOVA, and factor analysis are discussed with reference to the RCRAS' construct validity and the need for further extensive studies.