Fetal Alcohol Spectrum Disorder (FASD) is a general diagnosis for those exhibiting long-lasting neurobehavioral and cognitive deficiencies as a result of fetal alcohol exposure. It is among the most common causes of mental deficits today. Those impacted are left to rely on advances in our understanding of the nature of early alcohol-induced disorders toward human therapies. Research findings over the last decade have developed a model where ethanol-induced neurodegeneration impacts early neural circuit development, thereby perpetuating subsequent integration and plasticity in vulnerable brain regions. Here we review our current knowledge of FASD neuropathology based on discoveries of long-lasting neurophysiological effects of acute developmental ethanol exposure in animal models. We discuss the important balance between synaptic excitation and inhibition in normal neural network function, and relate the significance of that balance to human FASD as well as related disease states. Finally, we postulate that excitation/inhibition imbalance caused by early ethanol-induced neurodegeneration results in perturbed local and regional network signaling and therefore neurobehavioral pathology. Early ethanol exposure in humans can result in a range of measurable neurobehavioral deficits, clinically summarized as fetal alcohol spectrum disorder (FASD). FASD can be detected in afflicted offspring from early childhood with symptoms that often persist throughout adulthood. Reported characteristics of FASD include impaired cognitive performance, adaptive behavior disorders, deficits in sensory processing as well as executive functions of assessment and decision making, as well as a higher risk of seizures ,2,3,4,5.Neuroprotective agents hold a prophylactic potential for pregnant women diagnosed with alcoholism to significantly reduce the rate of children born with diagnosable FASD. However, potential drugs thus far found effective in limiting ethanol-induced neurodegeneration, such as lithium ,23,24,25There is a diverse array of assays for neural circuit function and dysfunction, with a wide range of spatial and temporal sensitivity. For example, single-unit electrophysiology allows examination on a millisecond time scale of the firing pattern of one or a few individual neurons within a selected brain region. In some cases, by analyzing firing patterns or by using juxtacellular labeling, the recorded cell can also be identified as a member of a specific cell class, such as a pyramidal cell or interneuron. Thus, a great deal of information regarding how an individual cell or cell class functions can be extracted with high temporal precision using single-unit techniques. However, it is increasingly apparent that neurons function in ensembles and networks, thus sampling an individual of a population may provide limited information regarding how a brain region functions as a whole. In contrast, techniques such as immunohistochemistry for immediate early gene products, such as c-fos, c-jun, arc, or zif allow assaying the activity of essentially all neurons within a brain region (or multiple regions), with resolution at the cellular level. These immediate early genes are “switched-on” when a neuron is activated, for example by sensory input, and the gene protein product can be detected by immunohistochemistry. When combined with double-labeling techniques, the identity of the activated cells can also be determined, for example as a GABAergic inhibitory interneuron or an excitatory pyramidal neuron expressing calcium-calmodulin kinase II. However, the tremendous spatial and cellular resolution comes at a cost. Immunohistochemical techniques provide individual snapshots of activity, and generally represent activity that is summed over many minutes. Thus, any temporal structure in the signal is lost.14C-2-deoxygucose uptake, functional magnetic resonance imaging (fMRI), or optical imaging of intrinsic signals) as indirect metrics of neural activity, share similar spatial/temporal trade-offs though generally with much reduced temporal precision than EEG or LFP recording. This trade-off between temporal and spatial resolution is apparent in essentially all functional neural assays. Electroencephalograms  and local field potential  recordings provide good temporal resolution but limited spatial resolution. Both of these techniques measure the activity of many thousands or millions of neurons relatively near the electrode. Given a similar number of active neurons, the greater the synchrony in activity of these neurons  the larger the recorded voltage fluctuations. Neural activity is metabolically expensive which provides another way to monitor brain physiology. Assays that measure metabolic activity or changes in oxygenated blood flow  and long-term depression (LTD) stimulation protocols.Finally, neural activity is ultimately dependent on synaptic transmission, and a variety of methods are available for direct assessment of relative synaptic strength. This can include either extracellular evoked potential recordings in simple monosynaptic circuits that follows direct electrical stimulation of afferent fibers, or intracellular recordings of spontaneous and evoked synaptic potentials/currents in individual neurons. These techniques can be performed either Any of the above techniques can be used to assay neural circuit function and detect changes from that function. All of these have been used to examine the long-term consequences of developmental ethanol exposure in humans and/or non-human animals including singe-units , local fThe analytical techniques described above can demonstrate disruption of normal neural circuit function following early ethanol exposure. In this section we briefly review what is known about how normal neural circuits work to encode information, and describe the roles of circuit components in said functions. This will provide a background for interpreting early ethanol induced changes in function and help identify critical circuit components impacted by early ethanol-induced dysfunction. In many regions of the brain, information is contained in both the spatial location of the active neurons  and the temporal structure of their spike trains. The brain processes and stores information in order to guide behavior. Nearly all sensory systems have some form of spatial organization related to the stimulus energies being processed, including retinotopic, somatotopic, tonotopic and odotopic maps in the visual, somatosensory, auditory and olfactory systems respectively. There may also be spatial organization of activity in the gustatory cortex based on the quality of the tastants producing a gustotopic map . SpatialThe ontogeny of spatial neural organization and spatial maps are heavily dependent on early experience. The formation and maintenance of neural circuits is activity-dependent, where active circuits are generally fortified and less active or less effective circuits are pruned. Furthermore, early experience can affect the structure and survival of both principal neurons and interneurons, thus even if neural circuit architecture was unaffected by early experiences, loss or impairment of interneuron function disrupting lateral inhibition could impair normal spatial functioning and acuity of the circuit.In addition to the identity or location of active neurons, information encoding is heavily dependent on the temporal structure of their activity. One form of temporal structure is an increase or decrease in activity, but beyond simple increases and decreases in firing rate of individual neurons, the patterning of neural activity between neurons is also important. This can easily be imagined in systems like motor control regions where specific muscle groups, controlled by specific motor neurons, must be activated in specific temporal sequences in order for the correct movement to occur. The same dependence on timing is true in all neural circuits. In addition to activity sequence, the synchrony of activity in disparate neurons is also critical to information processing. Temporal summation properties of neural dendrites means that the more synchronous pre-synaptic inputs are, the more effective they will collectively be in driving the post-synaptic neuron. An indication of neural synchrony can be gained from examining EEG and LFP recordings. The more synchronous neurons are within a region, the greater the sum of extracellular currents and resulting local potential. This activity often emerges as oscillations in the EEG or LFP. The strength of the oscillatory waves is influenced by the number of cells in the involved ensemble, the extent of synchrony in their activity, the number and strength of excitatory synapses connecting them, and the strength of synaptic inhibition. Oscillations can also be influenced by the biophysics of neural membranes, with some cells more tuned to specific frequencies than others due to the kinetics of the ion channels they express. Oscillations of this type have been described throughout the mammalian brain and often fall into a few bands of frequencies. Some of the more commonly studied oscillation frequency bands are delta , theta (5–15 Hz), beta (15–35 Hz) and gamma (35–90 Hz), though the specific frequency range labels vary between labs. Oscillatory activity has several consequences . First, The temporal structure of neural activity is an important factor in information encoding, information transfer, neural plasticity and the binding of disparate neural activity. Importantly, the balance of synaptic excitation and inhibition in a circuit plays a crucial role in shaping spike train temporal structure and network oscillations. For example, experimental manipulations of synaptic inhibition influence both the magnitude and the frequency of oscillatory activity. In the extreme, reductions in synaptic inhibition can result in epileptiform activity—a pathological oscillation.As described below, work from our lab and others have demonstrated that developmental ethanol exposure results in a long-lasting disruption of normal neural circuit function, assessed with many different techniques. Neural circuit dysfunction leads to impaired cognition and behavior. In this review we hypothesize that the long-term effects of developmental ethanol exposure on neural circuit function are due in part to a deficit in inhibitory interneuron development, and thus a disrupted excitatory/inhibitory balance. Disruptions in excitatory/inhibitory balance have also been recently hypothesized as major contributors to cognitive decline in Alzheimer’s disease , autism To examine early ethanol-induced effects on neuronal circuit function, rodent models have proven indispensible. Animal paradigms of ethanol delivery have developed over the past four decades toward dissecting the etiology of neurobehavioral deficits analogous to those observed in human FASD . Both chAcute ethanol effectively narrows the window of toxin exposure to a specific developmental period. When studying the direct effects of early ethanol exposure on brain development and long-term neuronal function, acute exposure minimizes many confounds inherent to a chronic exposure model. Specifically, protracted ethanol toxicity during chronic exposure cumulatively impacts other body systems that support brain development. For example, chronic alcohol exposure during gestation and lactation can negatively impact kidney function  and decrAcute ethanol exposure on the contrary provides a more discrete temporal window. A major drawback of acute ethanol exposure is its translation to a real-world model of FASD. Most acute “binge” exposure models generate an excessively high transient peak blood alcohol content often well above 200 mg/dL, which is already the equivalent of a medium build woman having over ten drinks in a five hour period . AdditioThe timing of each established FASD animal model has a neurodevelopmental equivalent to human gestation, which are not necessarily aligned by embryogenesic stage . The mosEffects of acute alcohol exposure can be examined during the earliest developmental stages by administering ethanol to the mother during gestation , or at a later developmental stage by direct administration to neonatal pups . Methods of ethanol delivery include water supplementation, ethanol vaporization, intubation/gavage delivery, or direct subcutaneous injection. In each case a peak blood alcohol concentration over 200 mg/dL is typically achieved, while 50 mg/dL is sufficient to elicit immediate activation of neurodegenerative cascades  with sigOne of the most notable effects of early ethanol insult is a devastating wave of neurodegeneration in the developing brain immediately following exposure. Multiple studies have reported a decrease in mass and volume of specific brain regions  and/or aThere are multiple implications for the long-term effects of anatomical changes of morphology and population numbers, because lasting deficits of neuronal circuit function and behavior in FASD models are likely due to early infrastructural changes in critically affected regions. It is important to consider how different brain regions and cell types are variably susceptible to ethanol-induced neurodegeneration when evaluating functional deficits characterized in FASD models ,35,63,69Interneurons have been shown to decrease in numbers in the long term when exposed to ethanol in the third-trimester equivalent of early postnatal development ,95,96; hin vitro studies have demonstrated how ionotropic ligand-gated ion-channels are targets of ethanol toxicity [Various assays of neuronal circuit function have been utilized to examine the long-term physiological impacts of early ethanol exposure, with the greatest emphasis on the hippocampus. Hippocampal structures are particularly sensitive to early ethanol binge-induced neurodegeneration and dysmorphology, and deficits in hippocampal-dependent memory are often a consequence. Appropriately, measurements of synaptic plasticity in the hippocampus have provided physiological confirmation of corresponding deficits in long-term potentiation ,72,105. toxicity ,107. Thitoxicity ,109. Interestingly, acute low-dose ethanol treatment has been shown to actually protect cortical neurons in culture from excitotoxicity under low concentrations of exogenous NMDA ,111. Addin vivo recordings of animal models with depth electrodes currently provide the most precise measurements of brain activity. We have recently reported significant long-lasting physiological effects in vivo, caused by binge-like ethanol delivered far earlier at postnatal day 7 [Two major foundations known to be impacted by ethanol: neuroanatomy and neurotransmission, are fatalistic determinants of long-term neuronal circuit function. This currently makes the precise etiology of ethanol-induced neuropathology a causality dilemma. As described above in Section-II, al day 7 . Both loal day 7 . Interesal day 7 . All defal day 7 . Given tal day 7 ,25,32,79al day 7 . In thisIn development of many forebrain circuits, early postnatal stages experience high excitatory activity and low inhibitory activity, setting a stage for a sensitive period of plasticity. P7 binge exposure occurs during this developmental stage for areas like neocortex and the olfacto-hippocampal circuit, and illustrates the importance of excitatory/inhibitory balance and the timely vulnerability of inhibitory progenitors in this FASD model. During this period, synaptic connections between excitatory neurons become structurally and functionally refined with progressively adapted inhibitory cooperation of surrounding interneurons, resulting in optimized excitation/inhibition balance and circuit function. Therefore, early alcohol insult can hinder the regular establishment of a functional circuit during the sensitive period in a way that is developmentally unidirectional and permanent. It has been hypothesized that artificially returning a circuit to early developmental stages of excitatory/inhibitory dynamics through various interactive and pharmacological interventions could allow a return to the sensitive plastic period for reassignment of otherwise faulty connections toward a more properly adapted circuit . Similar to our findings of long-term impairment in olfacto-hippocampal inhibitory activity following P7 ethanol , long-laEthanol is a GABA receptor agonist and acute ethanol exposure directly enhances GABA release from interneurons in neonatal neocortex while the ethanol is present . Howeverin vivo physiological measures becomes apparent here, because it may not always be possible to glean out behavioral changes within particular animal paradigms, yet there can be underlying disruptions in normal circuit function that are in fact significant. Such disruptions may be at the source of more complicated behavioral outputs currently carrying no observable animal model of behavior, but are particularly applicable to the nuanced neurobehavioral pathologies found in human FASD. In addition, pharmacological interventions guided toward FASD therapies can utilize in vivo physiological approaches to observe treatment effectiveness in real-time. With the advent of optogenetic methodology , in vivEach brain region, cell type and neuronal process impacted by ethanol exposure carries implications for subsequent measurable neurobehavioral deficits. The hippocampus is known to support spatial memory performance and experiences significant increases in neurodegeneration and decreased spine density after early ethanol exposure with a corresponding drop in spatial memory behavior task efficiency ,35,69,73There have been many observations of long-lasting deficits in sensory system-driven behavior in both animals and humans as a result of early ethanol exposure ,124,125.There is an increasing awareness that an imbalance in synaptic excitation and inhibition are associated with a variety of psychopathologies across the lifespan ,131,132,Imbalances between excitation and inhibition may be particularly deleterious during early development. Changes in synaptic inhibition are associated with the opening and closing of developmental sensitive periods ,142. ForComponents of social behavior in autistic children have recently been compared to those in children with FASD ,145. Addin vivo electrophysiology, reveal a disruption in local and regional activity in circuits that support behaviors coincidentally found to be impaired. Physiological alterations found in binge-exposure models of FASD involve decreases in inhibition at both local and regional circuit levels. As balance between excitation and inhibition is a critical foundation for normal neural circuit function, the disequilibrium observed in binge-exposure animal models of FASD illuminate a potential mode through which neurobehavioral deficits are generated. Imbalances  have been associated with a variety of developmental disorders including Alzheimer’s disease and epilepsy. Direct and indirect sensitivity of parvalbumin expressing GABAergic interneurons to a variety of insults maybe a common factor among these diverse disorders, and an important direct or indirect target of early ethanol exposure.Early developmental alcohol exposure has been shown to result in long-lasting neurobehavioral consequences. The precise etiology of reported deficits in animal models of FASD is by no means clear in any single paradigm. This is due to the multiple known modes of ethanol toxicity and the inherent complexity of brain development. Ultimate outcomes of early alcohol neuropathology, as measured by


Major depressive disorder (MDD) is characterized by a dysregulated fronto-limbic network. The hyperactivation of limbic regions leads to increased attention and processing of emotional information, with a bias toward negative stimuli. Pathological ruminative behavior is a common symptom of depressive disorder whereby the individual is unable to disengage from internal mental processing of emotionally salient events. In fact, lower deactivations of the neural baseline resting state may account for the increased internal self-focus. The insular cortex, with its extensive connections to fronto-limbic and association areas has recently also been implicated to be a part of this network. Given its wide-reaching connectivity, it has been putatively implicated as an integration center of autonomic, visceromotor, emotional, and interoceptive information. The following paper will review recent imaging findings of altered insular function and connectivity in depressive pathology. According to the World Health Organization, major depressive disorder (MDD) will be the second-leading cause of disability in the world by the year 2020, following heart disease  has garnered increasing attention as a dysregulated network in MDD. As the focus of this paper, the insular cortex, a region reported to be a part of the DMN and an important cortical structure implicated in emotional processing and regulation (among other functions) will be further examined in the context of MDD. Finally, recent imaging findings will be presented contrasting the role of the insula in depression across different information processing paradigms . As well, the insula’s role in the resting state and following therapeutic treatment will be examined.Emotional self-regulation is hindered in individuals suffering from MDD, causing a disruption in their attentional focus and creating memory biases toward negative life events. This results in an altered mood state due to negative sensory representations, which consequently modify cognitive processes  systems are clearly imbalanced, with substantial data indicating that NE responses are increased to stressor exposure and fearful stimuli, whereas the 5-HT system is hypo-responsive. These neurotransmitter alterations contribute to the over-activation of the amygdala, hippocampal, and cortical pathways activating stress and fear responsiveness  was observed in the insula, cingulate, and hippocampal brain regions in depressed patients compared to healthy controls  in depressed individuals in response to emotional events Drevets, , 2001. ADefault mode networks have further been investigated as impaired neural targets in MDD pathology. As mentioned previously, the brain’s “resting circuitry” involving cortical midline structures  serves as a neural baseline of task-independent activity, showing greater activation in response to non-demanding self-related processing [e.g., reflection and unconstrained mental activity (mind wandering); Kjaer et al., Using positive and negatively valenced pictures, Grimm et al. ,b demonsGrimm et al.  report hA recent meta-analysis by Delaveau et al.  reviewinParadigms exploring the brain at rest  provide a “state of the affairs” perspective of the neural baseline in MDD. To our knowledge, only two studies have explicitly examined insular activity in depressed populations using a resting state paradigm. The resting state in medication-free MDD patients has been shown to have a decreased functional connectivity in the left insula and amygdala with the resting state network , task-related paradigms aim to provide insight into the neural alterations associated with symptomatology in MDD patients. Videbech et al.  exploredUsing an autobiographical memory paradigm, Young et al.  examinedMajor depressive disorder patients also show impairments in adapting to different environmental or homeostatic states. Strigo et al.  evaluateMajor depressive disorder is associated with deficits in emotional processing, with a bias toward negative stimuli. This may be due to the fronto-limbic alterations in MDD pathology, whereby limbic regions are hypoactivated and frontal executive regions show decreases in activity. As such, the top-down modulation of emotionally based information is impaired, leading to depressive symptomatology . These deficits in emotional processing could be further exacerbated by the morphological changes  and neurochemical imbalances in the brain.Recently, much attention has focused on the neural basis of depressive pathology, particularly with respect to the baseline resting state in MDD patients. Disrupted connectivity in fronto-limbic structures is commonly reported as are abnormal deactivations in default mode regions. This supports the on-going mental processing of emotionally based information in MDD patients who have great difficulty disengaging from internal and external input. Of interest, the insular cortex with its many functions in emotional, sensorimotor and interoceptive processing, and extensive connectivity with DMN regions, also has been implicated in MDD pathology.Review of recent literature points to decreases in connectivity of the insula with the amygdala and default mode regions (anterior and posterior cingulate cortex). As well, the insula shows greater activity in the resting state, supporting the inability of MDD patients to disengage from externally cued events, which may lead to pathological self-focused mental ruminative behaviors. However, therapeutic brain stimulation studies have shown positive results in regulating insular activity by decreasing its activation in MDD patients, pointing to an effective therapeutic strategy in re-establishing functional emotional processing.Differing results have been obtained when looking at the processing of cognitive and emotionally based information in the insular region of MDD patients. Specifically, cognitive-based tasks requiring working memory, retrieval, and active processing showed decreases in the insula which may suggest impairments in integrating various cognitive processes required for mentally demanding activities. On the other hand, studies using emotionally based paradigms showed increases in insular activity. These findings may be due to the MDD patients’ greater susceptibility in attending to emotionally arousing stimuli. Indeed, greater insular activity was found primarily in response to negative emotions, pointing to impaired processing and regulation of negative mood and emotions.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Aequorea victoria in the early 1960s, bioluminescent proteins have greatly contributed to the design and initiation of ongoing cell-based clinical trials on cardiovascular diseases. In conjunction with advances in reporter gene technology, BLI provides valuable information about the location and functional status of regenerative cells implanted into numerous animal models of disease. The purpose of this review was to present the great potential of BLI, among other existing imaging modalities, to refine effectiveness and underlying mechanisms of cardiac cell therapy. We recount the first discovery of natural primary compounds with light-emitting capabilities, and follow their applications to bioanalysis. We also illustrate insights and perspectives on BLI to illuminate current efforts in cardiac regeneration, where the future is bright.Advances in bioanalytical techniques have become crucial for both basic research and medical practice. One example, bioluminescence imaging (BLI), is based on the application of natural reactants with light-emitting capabilities (photoproteins and luciferases) isolated from a widespread group of organisms. The main challenges in cardiac regeneration remain unresolved, but a vast number of studies have harnessed BLI with the discovery of aequorin and green fluorescent proteins. First described in the luminous hydromedusan  IntroductionMore than a century of bioluminescence: description of its molecular basisApplication of light-emitting proteins to bioanalysisImaging modalities in cell-based cardiac therapyLuminescent expression reporters illuminate cardiac regeneration effortsConclusions and future perspectivese.g. on summer nights, different species of terrestrial Lampyridae beetles, popularly known as fireflies, emit ‘flashes of light’ to find each other over long distances Bioluminescence is an amazing natural phenomenon, where light is produced by an organism; Stem cell–based therapies represent a promising treatment for cardiac damage, like that due to myocardial infarction. However, complete functional restoration has remained unsuccessful, despite intensive research in recent years in both experimental and clinical contexts e.g. magnetic resonance imaging (MRI) provides precise spatial resolution, whereas bioluminescence imaging (BLI) easily reports location and functional status. By using BLI, the activation of lineage-specific promoters driving the expression of photoproteins or luciferases can be used to track implanted cell behaviour. To date, BLI has been particularly useful in vitro where bioluminescence emission correlates linearly with emitting cell density, and in small animal models because there is limited loss or attenuation of a luminous signal due to the scattering phenomena when light must transmit across dense body tissues Application of distinct imaging modalities to overcome cardiac regeneration hurdles is basically at a developmental stage in animal models The purpose of this review was to present the great potential of BLI, among other existing imaging modalities, to refine effectiveness and underlying mechanisms of cardiac cell therapy before its clinical approach; we recount the first discovery of natural primary compounds with light-emitting capabilities and follow their applications to bioanalysis. We also provide insights and perspectives encouraging researchers to harness BLI to illuminate their current intensive efforts in cardiac regeneration.The first historical references to light-emitting reactions, including the introduction of the terms luciferin and luciferase, dated from 1885, when Emil du Bois-Reymond  mixed two different extracts from clams and beetles and produced light. This German scientist also found that one of the extracts was heat-sensitive, which led to the conclusion that there were at least two components in the reaction. The heat-sensitive chemical was hypothesized to be an enzyme, which he called luciferase; the heat-resistant compound was referred to as luciferin .in vitro light-emitting systems. Next, Strehler and Totter were the first to study the firefly reaction, which was not fully described until 13 years later. The firefly luciferase and luciferin structures were subsequently resolved by McElroy et al in 1956 and 1961 respectively.In recent history, numerous significant advances have been made in the biochemistry of bioluminescence Aequorea victoria, which produces a blue light. First, aequorin (blue light emitter) was discovered by Shimomura et al and, later, they also described the green fluorescent protein (GFP), which takes the blue light and shifts it to a green colour  when returns to the baseline conformation 2+, Mg2+ and ATP.Regarding the molecular basis of the bioluminescence reaction, the term ‘photoprotein’ refers to a primary reactant found in a specialized organ of an organism that is capable of emitting light in proportion to the amount of protein , and that is not the unstable, transient intermediate of a characteristic enzyme–substrate reaction Other important milestones in this field included: the elucidation of the mechanism of bioluminescence in many marine organisms (1967) Current research endeavours have increasingly demanded fast, portable, easy-to-use bioanalytical methods with a high level of functional integration. Bioluminescent reactants are quite versatile, sensitive tools with many attractive properties. They can be detected at extremely low concentrations; they avoid background interference from autofluorescent compounds typically present in biological samples; and they are compatible with many miniaturized platforms, like the lab-on-a-chip and lab-on-a-CD platforms. Thus, some of the above-mentioned light-emitting molecules have been used in a plethora of assays, including: intracellular assays for monitoring important biological molecules, like ATP and calcium; genetic assays involving regulation and detection; immunoassays; binding assays; and whole-cell biosensor assays, among others 2+-sensitive forms derived from coelenterates and jellyfish, was to measure and monitor calcium ions within various biological systems, mainly single cells 2+-activated photoproteins during cell activation or injury and provided a means to monitor protein synthesis in living cells One of the first applications of both photoproteins and luciferases, particularly the CaSubsequent bioluminescent analytical applications included quantifications of ATP The GFP, together with other fluorescent proteins with a wide colour range of emission (from blue to far-red) and versatile biochemical and photophysical properties, has also revolutionized the applications of molecular biology 2+ activity, monitor gene expression and track cells in intact living organisms Vibrio fischeri Y1 yellow fluorescence protein fused with bacterial luciferase hydroxyflavin. These chimeric proteins have yielded improved bioluminescent Ca2+ sensors for detecting intracellular Ca2+ changes in single cells, like neurons, zebrafish and mice Aequorea victoria jellyfish photoproteins (blue to yellow) was expanded to include other monomeric fluorescent proteins (orange to far-red) derived from non-luminous Anthozoa . In addition, new optical highlighters and Förster Resonance Energy Transfer (FRET) biosensors were developed. Thus, with these tools, we predict that any biological parameter can be investigated with an appropriate fluorescent protein–based application For example, it was initially recognized that protein labelling was feasible by producing chimeric proteins fused to GFP The harnessing of animals for research has been crucial for numerous medical advancements, including the development of effective treatments and the understanding of mechanisms involved in human disorders. Experimental procedures for testing regeneration in animal models typically require accurate characterization of implanted cell behaviour and functional benefits, including cell distribution, survival and differentiation status. Many existing analysis techniques are destructive; this necessitates replicating the experiments. Alternatively, bioluminescence offers the potential for non-destructive monitoring C; moreovThe first photonic detection in small mammals was performed in mice infected with bacteria that expressed luciferase proteins from a set of genes, the lux operon, which encoded the self-regulation and production of luminescent proteins The above-mentioned studies collectively exemplify the progress made in non-invasive imaging. This research has added new dimensions to our comprehension of several human diseases, including infection progression in vivo cell trafficking due to low background signal and high signal-to-noise ratio, but permit low spatial resolution.There are fundamentally three advancing non-invasive cell-tracking methods contributing to cardiac regeneration and translation to clinic: (1) paramagnetic labelling for MRI; (2) radioactive labelling for nuclear imaging, including positron emission tomography (PET) and single photon emission computed tomography (SPECT); and (3) reporter gene transduction for MRI, PET, SPECT, fluorescence and bioluminescence imaging in vivo implantation. Imaging of cells carrying reporter genes also holds promise for studying the subcellular basis of cell maturation and differentiation because they may be regulated under restrictive and lineage-specific promoters. In addition, multiple combinations of reporter genes can be applied into the same cell.Powerful cost-efficient optical imaging systems allow alternative high-throughput spatial and temporal monitoring with no radioactivity in the preclinical context. These exciting methodologies are based on the transfer of reporter genes, typically either bioluminescent or fluorescent, into cells. Successful reporter gene expression, which only occurs in viable modified cell, can be evaluated for quality control before Although the choice will depend on the intrinsic properties of each molecular-functional imaging tool, including availability and cost-benefit relationship as well as the biological process analysed, cumulative experience provides some foundations: (1) while MRI shows high resolution suffering from relatively low detection sensitivity, PET has both great sensitivity and resolution, but requires high-cost facilities; (2) bioluminescence and fluorescence procedures have excellent sensitivity, but relative depth penetration Ischaemic heart failure is the end stage of major cardiovascular diseases. It most often occurs when the blood supply to the heart is blocked. This leads to myocardial ischaemia and necrosis following the formation of a non-contractile scar and subsequent ventricular remodelling et al also studied the delivery of both cultured and freshly isolated adipose tissue–derived stem cells modified to co-express FLuc and GFP reporter genes following acute myocardial infarction in severe combined immunodeficient mice et al. used luciferase-based imaging to assess variable embryonic stem cell viability after transplantation into infarcted mice in vivo and ex vivo in mice and pigs respectively. In both models, meaningful functional benefits were also reported in vivoStem cell–based therapies are a promising strategy for regeneration of injured cardiac tissue, partially contributing to the generation of new myocardial tissue and vessels Basically, cell implantation approaches have included intracoronary delivery, direct intramyocardial injection or endomyocardial injection using specifically designed catheters in vivoEndothelial recovery is also a key challenge in cardiac regenerative medicine in vitro, luciferase-based cell labelling is not associated, e.g. with potential release of cytotoxic or pro-inflammatory particles when cells labelled with SPIO for MRI are being removed in vivoTaken together, this experience demonstrates that BLI is a valuable, unpolluted tool for the analysis of cardiac regeneration, will extend our understanding of the basis of optimal cell administration system and/or dose, and will greatly influence our views on the efficacy of future cell-based therapies. In comparison with other imaging modalities that also maintain cell viability, phenotype and differentiation capacity Although the evolutionary origins of bioluminescence remain obscure, light-emitting reactions include ecologically functional communication, courtship, attack-defensive and camouflage tools Aequorea victoria2+Aequorea victoria. Thus, these revolutionary discoveries were deservedly recognized in 2008, when Shimomura, Chalfie and Tsien were jointly awarded with the Nobel Prize in Chemistry for the discovery and development of GFP.From the first description of light-emitting aequorin and of its accessory protein GFP in the jellyfish Finally, the advent of reporter gene technology has also received much attention, because the light produced by photoproteins, luciferases and chimeras can be followed in single cells or in whole living organisms. This approach has promoted studies in gene expression, disease progression, and drug- or cell-based treatments of human multifaceted diseases. Presently, the major drawback of BLI is that it remains clinically unavailable focusing on animal models of diseases. However, new approaches have emerged for introducing specific mutations into genes that encode bioluminescent proteins; this has generated novel light-emitting reactants with different colour emissions


Tcrb) locus. These include transcriptional promoters and enhancers, which interact with each other in conformational space to form a promoter-enhancer holocomplex. The holocomplex subsequently recruits the SWI/SNF chromatin remodeling complex, leading to locus activation. However, the factors that mediate conformation and epigenetic changes required for efficient Tcrb gene assembly are unknown. We hypothesized that active transcription may stabilize the holocomplex. In order to test this, we chemically induced promoter-proximal-pausing in proT cells. We discovered that in the absence of elongating RNA polymerase II, the promoter-enhancer holocomplex is disrupted without any robust changes in transcription factor binding, structural protein recruitment or histone acetylation across the locus. Ongoing work seeks to decipher whether the transcriptional machinery is required at all for holocomplex stability. Meanwhile, in order to dissect the spatial and epigenetic mechanisms that lead to Tcrb activation, our lab has generated mutated versions of Tcrb cis-elements in a proT cell line model using Zinc Finger Nucleases. Preliminary studies on these mutants have shown that the holocomplex formation requires the enhancer (or enhancer bound elements) and also CTCF sites flanking the locus. Ongoing studies seek to identify which CTCF sites are necessary and sufficient for holocomplex stability. Using these and additional mutants, we are testing the role played by cis-elements in maintaining the chromatin and looping of Tcrb, which are essential for recombination.The adaptive immune system endows mammals with a sophisticated mechanism to survive in a world filled with pathogens. This dynamic defense is generated by V(D)J recombination, which enables lymphocytes to uniquely interact with an enormous range of epitopes on foreign antigens. V(D)J recombination is a set of sequentially controlled DNA cleavage and repair events, which brings variable (V), diversity (D) and joining (J) gene segments together to form functional antigen receptor genes. However, the recombination process must be stringently regulated to prevent formation of chromosomal translocations, which can lead to tumors. The process of V(D)J recombination is regulated at the level of tissue, stage and allele specificity by genetic and epigenetic mechanisms. Our lab has characterized several genetic elements that regulate chromatin accessibility and recombination at the T cell receptor beta (


The mechanical characteristics of endothelial cells reveal four distinct compartments, namely glycocalyx, cell cortex, cytoplasm and nucleus. There is accumulating evidence that endothelial nanomechanics of these individual compartments control vascular physiology. Depending on protein composition, filament formation and interaction with cross-linker proteins, these four compartments determine endothelial stiffness. Structural organization and mechanical properties directly influence physiological processes such as endothelial barrier function, nitric oxide release and gene expression. This review will focus on endothelial nanomechanics and its impact on vascular function. Endothelial physiology is influenced by numerous biochemical factors. Hormones, paracrines, autocrines and other mediators define the permeability of the endothelial barrier, the anti-thrombotic nature of the endothelial cell surface and endothelium-dependent blood pressure regulation , vessel wall tension, vascular hydrostatic pressure and cell–cell contacts determine the mechanical stimuli in the cardiovascular system. These stresses affect endothelial function via mechanotransduction, i.e., activation of mechanosensitive pathways Tzima . The corThe endothelial glycocalyx (eGC) is a thick carbohydrate-rich layer, lining the luminal side of the endothelial surface that consists of proteoglycans and glycoproteins. The proteoglycans are decorated with long carbohydrate side chains, the glycosaminoglycans, among which heparan sulfate is the most prominent in the eGC. This mesh serves as a host for specific plasma proteins, soluble proteoglycans and hyaluronic acid. Together, they form a dynamic and complex interface between blood and tissue Fig. . The totOne hallmark function of the eGC is the transmission of biochemical and biomechanical signals from the blood into endothelial cells. Changes in eGC nanomechanics can alter this function (=barrier function). Different processes are known that alter the nanomechanical properties of the eGC. As a polyanionic bio-gel, its volume and mechanics are regulated by the respective electrolyte concentration  stiffness in pulmonary endothelial cells and thereby increases barrier function. The barrier-enhancing effect is most likely mediated via a signaling cascade including cortactin activation and subsequent actin filament formation in the cell cortex physiological consequences in the control of endothelial permeability and the response to hormone action Hall . EndotheIn addition to chemical mediators, mechanical stimuli affect actin organization  and endothelial function. An in vitro increase in hydrostatic pressure, mimicking blood pressure in vivo, induces actin reorganization and affects endothelial permeability  span the whole cytoplasm. These fibers determine cell morphology and stability. They also contribute to focal adhesions and thus are involved in the mechanisms of cell motility (Pellegrin and Mellor Both microtubules and intermediate filaments determine cytoplasmic (bulk) stiffness Wang  Fig. 2)2).The molecular nature of the contribution of tubulin to cell nanomechanics has been investigated in detail  directly influence the flexibility of single microtubules, most likely resulting in a change in cellular elasticity  physiology is well documented and the molecular contributions to cell mechanics are known, evidence for a direct link between intermediate filament-dependent mechanics and endothelial function is still missing. Data on intermediate filament and microtubule mechanics and its impact on endothelial physiology indicate that several different nanomechanical signaling pathways exist that await future investigation.Each individual component of the cellular cytoskeleton described in the previous sections is directly linked to the cell nucleus. Plectin (nesprin-3) provides a link to the cytoplasmic network of intermediate filaments  anchoring of the mutated product in the nuclear envelope, results in a severe clinical manifestation termed Hutchison-Gilford progeria syndrome (HGPS)  glycocalyx, (2) cortex, (3) cytoplasm and (4) nucleus, are mainly determined by the composition of the respective structural elements. The dynamic interactions between those elements with cross-linker and motor proteins determine the mechanical properties of the respective region. Endothelial nanomechanics has a distinct influence on endothelial function. Various molecular mechanisms control the mechanical properties of living cells. In turn, cellular mechanics control intracellular signaling cascades. Thus, chemical and mechanical signaling pathways are strongly linked to each other. Nanomechanics provides information on the physiological state of the endothelial cell in terms of nitric oxide release and barrier function. A soft cell cortex, combined with a soft, well-hydrated glycocalyx, increases NO formation, which is a prerequisite for a functionally intact vasculature. Any changes in elasticity at the level of the eGC glycocalyx, the cell cortex and the cell nucleus, can have a significant influence on endothelial function in terms of local blood flow, tissue perfusion and, finally, arterial blood pressure.


This article provides a perspective on major innovations over the past century in research on the spinal cord and, specifically, on specialized spinal circuits involved in the control of rhythmic locomotor pattern generation and modulation. Pioneers such as Charles Sherrington and Thomas Graham Brown have conducted experiments in the early twentieth century that changed our views of the neural control of locomotion. Their seminal work supported subsequently by several decades of evidence has led to the conclusion that walking, flying, and swimming are largely controlled by a network of spinal neurons generally referred to as the central pattern generator (CPG) for locomotion. It has been subsequently demonstrated across all vertebrate species examined, from lampreys to humans, that this CPG is capable, under some conditions, to self-produce, even in absence of descending or peripheral inputs, basic rhythmic, and coordinated locomotor movements. Recent evidence suggests, in turn, that plasticity changes of some CPG elements may contribute to the development of specific pathophysiological conditions associated with impaired locomotion or spontaneous locomotor-like movements. This article constitutes a comprehensive review summarizing key findings on the CPG as well as on its potential role in Restless Leg Syndrome, Periodic Leg Movement, and Alternating Leg Muscle Activation. Special attention will be paid to the role of the CPG in a recently identified, and uniquely different neurological disorder, called the Uner Tan Syndrome. The Central Pattern Generator (CPG) for locomotion could hardly be introduced properly without describing first the spinal cord itself as well as the supraspinal and peripheral systems associated with its physiological basis.The spinal cord constitutes the most caudally located structure of the central nervous system (CNS). It is essentially a long and relatively thin neural structure that extends from the base of the skull  to the first lumbar vertebra. It is contained within the vertebral column but does not extend the entire length of that protective bone structure. In humans, it elliptical structure varies in length between 43 and 45 cm comprising 31 different segments (species-specific variations exist) – 8 cervical, 12 thoracic, 5 lumbar, 5 sacral, and 1 coccygeal segments. Every segment is associated with a pair (right and left) of spinal nerves. The spinal nerves comprise the sensory nerve roots (composed of 6–8 rootlets), which enter the spinal cord at each level, and the motor roots , which emerge from the cord at each level. In rostral parts, the spinal nerves exit directly from (just above from C1 to C7 or below from C8 and lower) the vertebra associated numerically with the corresponding spinal cord segment. However, in caudal parts of the spinal cord, the spinal nerves travel further down the column before exiting Netter, .In transverse sections, the spinal cord displays white and the gray matter tissues. Peripherally located, the former contains white matter tracts (ascending and descending myelinated fibers) containing both sensory and motor axons. Centrally located, the latter is characterized by its butterfly-shape that contains gray matter cells (unmyelinated) and, specifically, simple as well as more complex spinal circuits (see sections on reflexes and networks). In the center, there is a central canal that contains cerebrospinal fluids traveling up to the ventricles located in the brain. Peripherally, we find meninges that are layers of tissue surrounding the spinal cord for its protection – the dura, the arachnoid, and the pia. The latter is relatively thin and tightly associated with the surface of the spinal cord. The spinal cord is supplied by a vast system of blood vessels comprising mainly the anterior spinal artery, the bilateral sulcal branches, the bilateral posterior spinal arteries, the pial arterial plexus as well as the anterior/posterior spinal veins, the anterior/posterior sulcal veins, and the pial venous plexus . Ascending white matter tracts are mainly located dorsally and laterally whereas descending ones are mainly located ventrolaterally and ventrally. The ascending tracts convey sensory signals associated with the sense of touch, pressure, proprioception, and vibration via relatively large myelinated fibers . These tracts travel contralaterally (decussation) in the medulla prior to being redirected  toward the thalamus and eventually the sensory cortex  between the motor cortex  and the final common motor pathway, namely motoneurons in the spinal cord located at all segmental levels. Although often referred to as upper motor neurons, axons composing the pyramidal tracks have their cell bodies in the motor cortex and should accordingly and more appropriately be called corticospinal neurons. About¸ 80–90% of these corticospinal axons are known to decussate to the contralateral side at the pyramid level in the medulla oblongata. From there, they constitute the lateral corticospinal tract that sends input to spinal motoneurons in the ventral horn area. The remaining 10–20% descends ipsilaterally as part of the ventral corticospinal tract and decussates in the spinal cord prior to synapsing with the corresponding motoneurons . Some of the corticospinal neurons form instead the corticobulbar tract that sends input to brainstem motoneurons involved in the control of face, head, and neck muscles.An injury at this level is generally associated with the pyramidal tract syndrome characterized by spasticity, paralysis, and loss of skilled movements.On the other hand, cell bodies of the extrapyramidal tracts are mainly found in subcortical nuclei in the pons (reticular formation) and medulla oblongata. They send their axons in the spinal cord to motoneurons at all segmental levels. Specifically, the red, vestibular, tectum (superior colliculus), and reticular formation nuclei have cell bodies sending their axons through their respective descending extrapyramidal tract – the rubrospinal, vestibulospinal, tectospinal, and reticulospinal tracts. The former tract descends more laterally in the spinal cord whereas and the other three tracts travel ventrally (anterior column). The rubrospinal tract receives inputs from the motor and premotor areas 4 and 6. It sends projections to contralateral (decussation in the brain) motoneurons of the upper spinal cord segments for the control of both fine skilled movements and large powerful ones. It is generally considered to facilitate flexion and inhibit extension specifically in the upper extremities contralaterally. The tectospinal tract sends its axons in premotor lamina of the cervical spinal cord (VI–VIII) and is involved in neck and head motor control. In contrast with the other two tracks, the reticulospinal track sends projections to all levels of the spinal cord. It is involved in the control of autonomic functions . The vestibulospinal tract sends axons that travel either laterally or ventrally on the ipsilateral side of the spinal cord to premotor laminae VII and VIII. It is known to control muscular contraction levels specifically in extensors. The olivospinal tract originates in the inferior olivary nucleus and innervates contralaterally segments in the spinal cord.Lesioned extrapyramidal tracts are associated with the extrapyramidal syndrome characterized by involuntary movements, muscular rigidity, and immobility without paralysis. Most of the descending tracts described above have roles to play normally  in inducing and modulating stereotyped motor behaviors such as locomotion  and ascending (sensory-related) information between the brain and spinal cord. In recent years, spinal cord physiologists have undertaken to characterize in greater details the cellular organization and function of neurons of the spinal gray matter. While it has been known for years now that some of those neurons form local spinal reflex pathways , others have been more recently identified as part of relatively complex circuits involved directly in the control of stereotyped motor behaviors – specifically locomotion. Other rhythmic motor behaviors associated with spinal cord networks  remain poorly characterized at least cellularly , two (disynaptic), or more (polysynaptic) synapses and corresponding neurons . Simple reflexes may be of autonomic  or somatic  origin. The latter system has been more extensively studied mainly in cats. Among the known somatic spinal reflexes, the Ia, Ib, II, FRA (flexion reflex afferent) reflexes have been particularly well-characterized although their roles in the control of locomotion remain incompletely understood.The so-called Ia monosynaptic reflex arc constitutes the best known somatic reflex pathway. It is also considered the simplest and fastest reflex of all. It mediates primary afferent (Ia) inputs from muscle spindles typically activated by muscle stretch . The Ia afferent input enters the spinal cord  and establishes monosynaptic connections with homonymous alpha-motoneurons  in the gray matter . This excitatory reflex is known to increase homonymous muscle contraction in response to muscle elongation. In addition to monosynaptic connections with the corresponding homonymous muscle, Ia afferents possess branches that also establish monosynaptic connections with synergistic alpha-motoneurons . It is generally considered to play a role in tonus and postural adjustments. Although originally believed to be “non-modulatable,” evidence in recent years has shown that activity levels in the Ia monosynaptic arc can vary depending upon circumstances, phases, and tasks – e.g., amplitudes of the H-reflex  decrease during walking compared with standing quietly. Also, during walking, H-reflex amplitudes are typically smaller during the swing phase (flexion) than the extension phase . It has been described initially as inhibiting the homonymous and synergistic alpha-motoneurons at rest although this specific inhibitory action may remain quiescent  in other conditions such as during locomotion. Inputs from the Ib afferents enter the spinal cord and inhibit disynaptically  alpha-motoneurons at rest. This inhibitory reflex was originally believed to act as a protective mechanism against excessive muscle contraction  since FRA stimulation under experimental conditions was shown to reset the step cycle to flexion  pathway activated by relatively high-threshold fibers . Ipsilaterally, it involves at least two interneurons (three or more synapses) in several segments of the spinal cord and alpha-motoneurons of several flexor muscles. It is also called the nocifensive reflex or withdrawal reflex pathway. Specifically, it comprises at least two excitatory interneurons (three synapses) for ipsilateral flexor motoneuron activation and two interneurons (one excitatory and one inhibitory) for ipsilateral extensor motoneuron inhibition. If the stimulus is particularly strong and intense, it may be accompanied by an activation of contralateral pathways and, thus, lead to the crossed-extension reflex producing concomitantly an extension of the contralateral limb or leg. Additional interneurons (and decussation) are involved in mediating the crossed-extension reflex – two excitatory interneurons for contralateral extensor motoneuron activation and two interneurons (one inhibitory and one excitatory) for contralateral flexor muscle inhibition. These reflex pathways are thought to play a role in withdrawal of a limb  from a painful stimulus whereas the crossed-extension reflex would serve to enhance postural support during withdrawal of the affected limb from the painful stimulus. Task-dependent modulatory responses have also been found during locomotor activity since a long-lasting burst of activity is unraveled ipsilaterally during pharmacologically induced fictive locomotion (long-lasting FRA response). The corresponding interneurons have even been proposed to be part Although additional pathways have been associated with other “simple” reflex acts , it is beyond the scope of this review to describe them all. Suffice to say that clinically, dysfunctional reciprocal inhibitory reflex pathways have been associated with increased co-contraction found in different pathological conditions such as Parkinson’s disease and spasticity.To date, the best-characterized spinal network is undoubtedly the CPG for locomotion that directly controls the basic motor commands underlying ambulation. Originally believed to be of peripheral origin (former conclusion of Sir Charles Sherrington), it has been subsequently shown by his former student, Thomas Graham Brown, to be centrally located and composed of different sets of spinal interneurons  of the spinal cord. That was elicited specifically when dropping one of the limbs from a flexed position  could participate in the production of that locomotor pattern by receiving strong excitatory input from FRA interneurons given their corresponding rhythmic activity in l-DOPA-treated TX cats.However, it is only with the development of intracellular recordings that, in the 1960s, came the first evidence cellularly of its existence . Indeed, with its simpler neural system, it has been easier to investigate extensively neuronal activity from single spinal cells using the in vitro isolated rodents  as well as, subsequently, in in vivo TX rodents (adult rat and mouse models) led to substantial advances in cellular target identification (receptors and channels) associated with locomotor rhythm-generation and modulation. Blood brain barrier permeable ligands have served to pharmacologically “dissect” in vivo the contribution of specific receptors and channels to locomotor rhythm-generation. Clear CPG-activating effects induced by specific drugs have also been found in various in vitro preparations from invertebrate and vertebrate species, although it is beyond the scope of this review to report on all of them  have largely contributed to understand and further describe the detailed organization of the locomotor CPG. Experiments in l-DOPA has been among the first systems to be associated with CPG activation in acutely TX cats and rabbits .As mentioned earlier, the noradrenergic system and specifically l-DOPA, serotonin (5-HT) or dopamine (DA) receptor ligands such as 8-OH-DPAT, buspirone (5-HT1A/7 agonists), quipazine (5-HT2A/2C agonist), and SKF-81297 (D1-like agonist) were found to trigger significant locomotor-like movements  whereas others ligands such as TFMPP (5-HT1B), m-CPP (5-HT2B/2C), SR57227A (5-HT3), or clonidine  were shown to elicit mainly non-locomotor movements  in TX mice .Using selective antagonists and genetically manipulated animals , it has been clearly established that NMDA, 5-HT1, 5-HT7, 5-HT2A, and D1 receptors were specifically involved in mediating such CPG-activating locomotor-like effects  remains unclear (see section below). However, dual immunohistochemical experiments recently showed locomotor activity-labeled (c-fos) 5-HT1A-, 5-HT2A-, or 5-HT7- positive neurons in the cat lumbar cord  suggesting that some of the locomotor activity-related receptors identified recently in in vivo models may indeed be located on CPG neurons.Subsequent studies conducted in our laboratory have shown that only simultaneous activation of some of these candidate receptors can, using similar pharmacological approaches and animal models, induce full locomotor-inducing effects. Partial CPG-activating effects  induced by some ligands, as mentioned above, were found indeed to turn into full CPG-activating effects  by simultaneously administrating 8-OH-DPAT, quipazine, Guertin, , 2009a. in vivo models of SCI . However, it has remained difficult to consider those results as evidence of cellular targets associated with CPG activation or modulation given the used paradigm . One of them is the population of V0 interneurons that was associated with left-right alternation since mice without V0 interneurons  displayed bilateral synchrony rather than bilateral alternation during locomotion  may constitute different cellular components of the CPG. For instance, V0 interneurons form many cell types including commissural neurons which could establish inhibitory reciprocal connections between the two sides of the spinal cord. In turn, V1 interneurons may be associated with inhibitory interneurons such as the Ia inhibitory and Renshaw cells whereas HB9s may be excitatory interneurons constituting at least part of the rhythm-generating layer. However, additional studies need to be conducted in order to fully characterize these promising new CPG neuron candidates . Plateau potential is another intrinsic property believed by some researchers to be associated with endogenous (tetrodotoxin-resistant) pacemaker-like activity  Ca2+ channel, persistent Na2+ current, Ih current, IA current, ICAN, INaP current, IK(Ca) current, post-inhibitory rebound, reviewed in Harris-Warrick, There have been indeed several cell populations in the corresponding area of the CPG  shown in the last few decades to be capable of expressing pacemaker-like properties in specific conditions such as in the presence of NMDA alone or combined, at lower dose, with serotonin and DA  and/or pharmacological substances  can not be considered as valuable evidence for the existence of a CPG in humans given that improvement may be attributed largely to modulation of simple local reflex arcs activated cyclically during training This section could in itself constitute a separate review article given the substantial body of data existing on that matter. Only a brief overview will be presented here.First of all, the role of primary afferent inputs during locomotor activity is incompletely understood. However, it makes no doubt, in non-pathological cases, that the control system requires those inputs for successful progression in real life conditions. This said, results obtained from TX and deafferented animal models have clearly shown that the spinal cord is capable of generating rhythmic locomotor-like output and corresponding movements in the absence of descending and peripheral signals  may sometimes contribute to impair the remaining walking capabilities whereas, in other case, spasticity is believed helpful to promote or trigger the swing or flexion phase  in animals and humans constitute a clear sign of such phenomenon  of high-thoracic or cervical TX rats  in L1–L2 segments may possibly be considered as among the first cellular events associated with cell property changes and increased CPG excitability post-SCI  were found to display up- or down-regulated expression levels in sublesional spinal cord areas within a few hours to a few days post-trauma. Landry et al.  reported1A/7 receptor expression in lumbar spinal cord segments of chronic paraplegic cats  and preprodynorphin because their expression was found to display increased levels in lumbar segments several hours to several days post-TX , several signs, or pathological complications associated with rhythm-generator problems have been reported over the years. For instance, nystagmus or involuntary rhythmic eye movement is an ocular problem that is associated with heredity problems, poor vision, cortical blindness, intracranial neoplasms, or cerebellar ataxia. Pending upon the specific cause, different types of treatments and corresponding rhythmicity or “pacemaker” problems have been proposed  and lower (lumbar) locomotor CPGs has been shown in humans to undergo a decrease in coupling strength during childhood and the development of bipedality. The shared or coupled CPG hypothesis has been first postulated by Grillner  to explaAll in all, given these evidence and findings, it is not unreasonable to postulate that impaired equilibrium that may be directly attributed to cerebellar atrophy and deficits in UTS patients could have enabled the expression of more tightly coupled activities between the upper and lower CPGs for the development of quadrupedal walking instead of bipedal locomotion.www.NordicLifeSciencePipeline.com). If safety data are found, it will be of interest to eventually explore and develop such a treatment also for bipedal training of UTS patients. As discussed earlier in the review, several afferent pathways and simple reflex arcs have been shown to modulate stepping in real life conditions. This will perhaps require additional adjustments and specific pharmacological aids to separately control spasticity and other peripheral input problems that can impair normal bipedal ambulation in some pathological conditions , corresponding sublesional plasticity changes may have occurred as a compensatory mechanism for preserving, at least, the expression of some form of CPG activation and walking capabilities. Plasticity changes that may be found are probably similar, to some extent, with those described in animal models of SCI . Ancestral upper CPG activity, enabled or uncovered by this pathological condition, would not restore equilibrium but at least some descending drive  capable to support some leg movements during progression with all four extremities. Based upon these hypotheses, the next question is: Can specific therapies be proposed for restoring normal CPG coupling and activity and corresponding bipedal walking in UTS patients? Although unlikely to be proposed in a near future as a standard therapy, it is obvious that cell replacement and grafting approaches that can repair the cerebellum of UTS patients would constitute an ideal solution for the recovery of bipedal walking. In the meantime, other approaches, strategies, and cellular targets could possibly be proposed and explored. One of them is obviously associated with means that can improve vestibulospinal control over extensor activity in the lower limbs. Electrical stimulation of extrapyramidal tracts  or corresponding nuclei for improving or restoring posture and equilibrium capabilities, as shown in cats  and could still be explored (UTS) to restore normal CPG activity and corresponding bipedal walking capabilities. The CPG for locomotion has clearly been shown to be both flexible and adaptable (e.g., Harris-Warrick, The author wishes to declare financial competing interests in Nordic Life Science Pipeline.


Size distributions had not changed significantly but large corals had declined over 20 years. Thus, data from a wide range of sites taken over two decades support claims by others that climate change is indeed beginning to show clear effects on Red Sea reefs.Three independent line intercept transect surveys on northern Red Sea reef slopes conducted in 1988/9 and 1997/8 in Egypt and from 2006–9 in Saudi Arabia were used to compare community patterns and coral size. Coral communities showed scale-dependent variability, highest at fine spatial and taxonomic scale (species-specific within and among reef patterns). At coarser scale (generic pattern across regions), patterns were more uniform . Neither fine- nor coarse-scale patterns aligned along the sampled 1700 km latitudinal gradient. Thus, a latitudinal gradient that had been described earlier from comparable datasets, separating the Red Sea into three faunistic zones, was no longer apparent. This may indicate subtle changes in species distributions. Coral size, measured as corrected average intercept of corals in transects, had decreased from 1997 to 2009, after having remained constant from 1988 to 1997. Recruitment had remained stable (∼12 juvenile corals per m Climate change is a major threat to the future of the world's coral reefs th century and assessment and monitoring work since the 1970s We sampled along a 1700 km gradient from the Gulf of Suez with some of the northernmost reefs in the western Indo-Pacific, to the Farasan Islands of tropical latitude in southern Saudi Arabia . The RedIn the present paper, we evaluate several datasets obtained along the length of the Red Sea by the same researchers over the span of two decades. This rich dataset allows exploration of patterns driven either by spatial or temporal determinants. We examine characteristics of areas sampled at different times, different latitudes to see whether described gradient and patterns Stylophora pistillata). In the Farasan Islands, Farasan Banks and the Yanbu area, predation by the crown-of-thorns-starfish  had denuded some reefs prior to sampling and only very few, or single, species and/or colonies were encountered . Species dominance of surviving corals reflected the taste of the predator Analyses included 193 transects from a total of 63 sites grouped in four regions  that werPorites lutea , Stylophora pistillata , P. (Synaraea) rus , Millepora dichotoma (dominant in semi- and current-exposed areas everywhere) and Acropora cytherea (dominant on wave-exposed reef-slopes) in Saudi Arabia, A. hemprichii and A. valida in Egypt. Within each sampling area, the composition of locally dominant species varied  showed consistency at optimal clusters level determined by maximizing the cophenetic index. No separation of samples into unique clusters according to year of sampling occurred, suggesting temporal consistency of data and absence of a primarily temporally-determined pattern . In all analyses, clusters included transects from all sites  and no rAcropora slopes and leeward Porites slopes and coral carpets, as previously described from the region Millepora-dominated sites and sites of mixed dominance had greater mean depth than the Acropora and Porites dominated clusters, but differences were not significant.Analysis based on presence/absence data showed no separation of any site groups based purely on latitudinal position or year of sampling, suggesting that these were not the main factors determining variability. Use of the same clustering algorithm as in The pattern of clustering suggested high variability of community pattern on a fine scale  but absence of a consistent gradient across the entire study area. This means that coral communities in similar setting on nearby reefs could be characterized by a variety of species, but that most of these species-specific community differentiations were not unique to any region. Where unique communities were found, these only occurred in specific environments alongside more widespread community types on nearby reefs. On a coarser, generic level resolution, our dataset suggested more uniformity of pattern and the presence of relatively homogenous differentiation throughout the entire study region. In combination, these findings hint at the potential loss of regionally unique patterns as previously observed by Comparisons of summary statistics between sampling areas and years show absence of statistically significant differences in either coral cover, untransformed coral intercept length as a proxy for coral size distribution, diversity or dominance indices . There wP. (Synaraea) rus and P. columnaris coral carpets , since corals in the largest size-classes had always been rare  showed high coral cover on dense een rare .2. Absence of change in coral size distributions among surveys  per sampling unit, suggesting 13 juvenile corals per m surveys  may alsoResults from three reef assessments in the Red Sea over two decades suggest temporally consistent and scale-dependent community variability  with overall little ordering along a latitudinal gradient as well as a decline in average coral size. While the former suggests potentially system-wide changes in comparison to previous findings Our datasets were internally consistent, since all surveys were executed and evaluated by the same person using the same method (10-m-long line intercept transects). The adequacy of transect length had been tested S. pistillata) and some deep Farasan Island transects (dominated by A. clathrata and A. downingi that do not occur in the N). Thus, while the present dataset found evidence for regional variability, a clear picture of absence of an ordered latitudinal gradient of community pattern emerged. These results are compatible with patterns observed along a similar spatial gradient in the Great Barrier Reef While P. nigrescens, A. grandis). This may suggest that some of the rarer coral species, some of which were responsible for the latitudinal gradient observed by A. muricata, A. nobilis, Montipora stellata recorded by Like elsewhere, Red Sea coral reefs have been subjected to serious disturbances over the past few decades. The 1998 bleaching event affected reefs in Saudi Arabia Acropora on the more exposed sides, Porites in lagoons, more sheltered areas and in many coral carpets, and Millepora at current-exposed sites. This is in accordance with observations in the literature The most common, zone-forming genera and species showed consistency throughout the sampling area. On upper reef slopes (<12 m), the typical dominant corals were P. lutea and medium deep P. (Synaraea) rus and P. columnaris carpets that contained exceptionally large and healthy colonies in the 1980s and early 1990s Previous studies in the Gulf of Aqaba In conclusion, our study points to potentially increasing community homogenization and decline in average size of coral colonies throughout the Red Sea. These are phenomena that have also been observed in other peripheral seas. Different to the Caribbean, where community homogenization results from loss of dominant species and replacement by novel, but region-wide occurring communities 2). A line down the center was then taken as the transect-line equivalent and intercepts of all encountered corals were measured to the nearest mm. All transects were recorded and evaluated by the same author (BR), eliminating observer-specific identification or measurement variability. Coral cover information from sites between Hurghada and Ras Banas obtained by visual estimation of cover in 2×2 m sampling squares in 1997 were also used for comparison. Adequacy of transect length and quadrate size had been tested previously Coral communities were sampled on Egyptian and Saudi Arabian offshore reefs between the Gulf of Suez and the Farasan Islands, a latitudinal gradient of ∼1700 km, by means of 10m-long line-intercept transects D, approximated as non-overlapping circles of an unknown distribution in the plane) using the harmonic mean of the chord lengths y . Also cumulative intercepts per species can be a useful measure. If intercepts were to markedly decrease among surveys, this, together with calculation of D (eq. 1) could be counted as indication for decline of large corals. Both approaches were used to evaluate stationarity or change of average coral size. Size distributions of intercepts were corrected for bias following For comparison of community pattern based on relative coral cover per species/genus, all individual coral intercepts were pooled per taxonomic unit. To evaluate colony sizes, a dataset of intercept measurements for every single encountered coral was produced. Coral intercept is not a simple measure of coral size (diameter), since neither the position of intercept on the coral, nor the distribution function of either the intercepts or of the underlying coral diameters are known. From the datasets of individual coral intercepts, considering them chord-lengths intercepting circles in the plane, the relationship developed by Species names were harmonized between surveys to account for different taxonomic interpretations Coral recruitment was evaluated from the unbiased


Prominent neuroscience models suggest that addictive behavior occurs when environmental stressors and drug-relevant cues activate a cycle of cognitive, affective, and psychophysiological mechanisms, including dysregulated interactions between bottom-up and top-down neural processes, that compel the user to seek out and use drugs. Mindfulness-based interventions (MBIs) target pathogenic mechanisms of the risk chain linking stress and addiction. This review describes how MBIs may target neurocognitive mechanisms of addiction at the attention-appraisal-emotion interface. Empirical evidence is presented suggesting that MBIs ameliorate addiction by enhancing cognitive regulation of a number of key processes, including: clarifying cognitive appraisal and modulating negative emotions to reduce perseverative cognition and emotional arousal; enhancing metacognitive awareness to regulate drug-use action schema and decrease addiction attentional bias; promoting extinction learning to uncouple drug-use triggers from conditioned appetitive responses; reducing cue-reactivity and increasing cognitive control over craving; attenuating physiological stress reactivity through parasympathetic activation; and increasing savoring to restore natural reward processing. Treatment and research implications of our neurocognitive framework are presented. We conclude by offering a temporally sequenced description of neurocognitive processes targeted by MBIs through a hypothetical case study. Our neurocognitive framework has implications for the optimization of addiction treatment with MBIs. Although addiction has been a subject of societal concern for millennia, over the past several decades, discoveries from cognitive and affective neuroscience have deepened our understanding of this age-old, vexing, and pernicious problem. The perspective emerging from several lines of research is one in which addiction is seen as a cycle of compulsive drug-seeking behavior fueled by dysregulated neurocognitive processes , 2. Key Despite these advances in the basic science of addiction, many behavioral interventions for this disorder lag behind the ever-accelerating pace of discovery and have yet to systematically integrate and apply neuroscience findings into treatment development. To the extent that novel psychological therapies target dysregulated neurocognitive processes underlying addictive behavior, they may hold promise as effective treatments for persons with substance use disorders. Mindfulness-based interventions (MBIs) such as Mindfulness-Based Relapse Prevention , 5 or MiThis review offers a novel conceptual framework with which to understand how MBIs may ameliorate addiction, with a focus on how such interventions target pathogenic cognitive, affective, and neurobiological mechanisms that contribute to addictive behavior. This framework is first grounded in a brief description of risk chain linking cue-reactivity, implicit cognition, and dysfunctional cognitive control efforts that drives the appetitive motivational states and drug-seeking behaviors characteristic of addiction. Next, we propose a number of hypothetical neurocognitive targets of MBIs, and critique supporting empirical evidence from extant literature on mindfulness and addiction. To promote future research in this area, we recommend behavioral tasks and psychophysiological measures that could be used as mechanistic probes of MBI treatment effects. Finally, we suggest how our neurocognitive framework might lead to optimization of the next generation of MBIs for persons suffering from addictive disorders.Research with animals and humans demonstrates that chronic administration of psychoactive drugs results in adaptations in multiple neurotransmitter systems in the brain, consequentially altering functional neural circuitry that governs a broad array of interactive processes . Though the neurobiological bases of the effects of chronic drug abuse on brain-behavior relations remain to be fully elucidated, current research suggests that the dopamine system plays a critical role in the progression from drug initiation to chronic and habitual self-administration. Psychoactive drug-use induces dopaminergic activity in the ventral striatum and ventral tegmental area , resultiRepeated use of psychoactive substances is believed to impart motivational significance to cues associated with drug-use episodes through sensitization of mesocorticolimbic brain regions . As suchThis conditioned response to drug-related cues , manifests as a constellation of somatic sensations coupled with a broad array of physiological reactions including autonomic, corticolimbic, corticostriatal, and neuroendocrine responses –19. The Because obtaining and consuming psychoactive substances are motivationally salient goals in addiction, drug-use action schemas stored in memory guide implicit cognitive processing of stimuli associated with previous drug-use episodes. This implicit cognitive process is manifested as a preferential focus of attention toward drug-related cues, known as addiction attentional bias . When atAs the addictive habit becomes more entrenched, individuals struggling with addiction experience a loss of cognitive and behavioral control. Cognitive control deficits include those affecting attentional and inhibitory control  and defiAs a result of these cognitive control deficits, actively addicted persons may experience an intense, overwhelming compulsion and motivation to seek and use drugs that is difficult to regulate. Moreover, persons in recovery from addiction often experience the impulse to use substances as intrusive and incongruent with their desire to remain abstinent . Yet, duThis failure to resolve conflict between emotional drives  and higher-order goals  during attempted suppression may further bias cognitive processing toward drug-related cues and mental contents, inadvertently increasing attention to substance-related thoughts and urges , 51. ConIn sum, the maintenance of drug addiction is a manifold process, putatively subserved via dysregulation within and between multiple, dynamic neurocognitive processes. Though not an exhaustive model of the time-course of addiction, we focus here on three primary systems where neuroscience models of addiction from the extant literature converge to shed light on dysregulated behavioral control: 1) habit responding-automaticity  behavior. From this neurocognitive perspective, addiction occurs through basic human learning processes gone awry; neural resources devoted to learning become hijacked due to the neuropharmacologically rewarding properties of the substance . Once anAlthough mindfulness is an English term linked with a set of contemplative practices and principles originating in Asia over 2500 years ago, in its modern usage, mindfulness refers to a psychological phenomenon currently being studied for its relevance to mental and physical health in fields such as medicine, psychology, and neuroscience. Across these fields, a body of literature has accrued supporting the efficacy of MBIs for a range of biobehavioral disorders, including but not limited to addiction. Indeed, there is support for the effectiveness of MBIs in reducing stress and improving clinical outcomes across disorders as diverse as depression , irritabstate of mindfulness, a mindset characterized by an attentive and non-judgmental metacognitive monitoring of moment-by-moment cognition, emotion, perception, and sensation without fixation on thoughts of past and future . These processes do not operate in isolation; they are linked in mutually interdependent, interpenetrating, recursive networks . MBISubstance dependent individuals typically experience euphoria during initial stages of drug-use. Yet, as experience with the drug increases, the reward associated with drug-taking becomes dramatically attenuated. Despite diminishing returns in positive emotional experiences resulting from substance use, dependent users continue to use their drug of addiction. Undergirded by neuroplastic changes in striatal circuitry, habitual drug-use becomes an overlearned process that can become automatized , 80.Relatedly, human positron emission tomography (PET) research has found that meditation practice increases dopamine release in the ventral striatum . This piThe practice of mindfulness in daily life is focused on developing awareness of automatic behavior. Indeed, many MBIs prescribe informal mindfulness practices where individuals are instructed to engage in everyday, repetitive tasks  with full consciousness of the sensorimotor aspects of the activity. Such informal mindfulness practices are designed to reduce mind-wandering and strengthen conscious control over automaticity. Potentially as a result of such practices, mindfulness training has been shown to decrease habit behavior  and reduGiven that drug-use action schemas may be evoked by cues associated with past substance use episodes, activation of addictive habits may be interrupted by re-orienting attention from substance-related stimuli to neutral or salutary objects and events. MBIs may be especially efficacious in that regard. Focused attention and open monitoring mindfulness practices capitalize on attentional orienting, alerting, and conflict monitoring – the fundamental components of attentional control , 90. ConMBIs may target addiction attentional bias by facilitating attentional disengagement from substance-related stimuli. In support of this hypothesis, a study of alcohol dependent adults in residential treatment identified a significant negative correlation between dispositional mindfulness and alcohol attentional bias for stimuli presented for 2000 ms that remained robust even after controlling for alcohol dependence severity, craving, and perceived stress . HypotheHence, mindfulness training may strengthen the capacity to regulate attention in the face of conditioned stimuli associated with past substance use, countering attentional biases by refocusing attention on neutral or health-promoting stimuli . Repeatedly redirecting attention from substance-related cues toward innocuous stimuli may foster extinction of associations between substance-related cues and drug-use action schema. This potential mechanism may explain how attentional bias modification among addicts leads to decreased substance use and improved treatment outcomes , 101. FuThe urge to seek intoxication from addictive substances is driven, in part, by reactivity to substance-related stimuli which have been conferred incentive salience, and is magnified by negative affective states. Several studies demonstrate that MBIs can produce significant reductions in craving , 102–105Mindfulness-based interventions may positively influence craving-related processes in several ways. First, mindfulness training may decrease bottom-up reactivity to drug-related stimuli, as mediated by reduced activation in the subgenual anterior cingulate cortex and striatum during exposure to substance cues . Second,MBIs may also produce therapeutic effects by increasing awareness of implicit craving responses. Tiffany  proposedwhich cues are under the spotlight of attention, and become more sensitive to how those cues may trigger changes in body state and motivation drive.In synthesizing the findings regarding attentional bias and cue-induced craving, we suggest that MBIs may restructure attentional bias away from drug-related reinforcing stimuli  and facilitate the addict’s attempts to deal with associated cravings. We posit that mindfulness-centered regulation of cue-elicited appetitive responses occurs as a result of strengthening frontal-executive circuit-function and enhancing neural communication to the hippocampus and thalamus through formal and informal mindfulness meditation practices. The hippocampus is critical for context-dependent learning and memory – with reciprocal connectivity to brain regions that code for reward , interoception (insula), affect , and thalamus. In turn, the thalamus, a complex structure that is generally considered to serve as a relay station between limbic, striatal, and cortical circuits, contains efferent and afferent projections with striatal, limbic, somatosensory, ACC, lateral and medial PFC, and OFC. Thus, the recovering addict may utilize mindfulness training to become aware of Hence, mindfulness may increase awareness of craving and thereby facilitate cognitive control of otherwise automatic appetitive impulses. In that regard, a recent study found that participation in Mindfulness-Oriented Recovery Enhancement was associated with decreased correlation strength between opioid craving and opioid misuse, suggesting that mindfulness training may have decoupled appetitive responses from addictive behaviors . This meThe effects of mindfulness on cognitive regulation of craving might be measured by utilizing neuroimaging methodology  to investigate neural circuitry function while participants attempt to regulate their craving response to salient drug-cues. For example, cognitive regulation appears to decrease cigarette craving concomitant with increased activity in dACC  and prefInsofar as stress evokes automatic responses and impairs prefrontally mediated cognitive control functions , exposurIn contrast, mindfulness, which has been conceptualized as a non-reactive form of awareness  may enabOne approach to evaluating the effects of mindfulness-centered regulation of stress appraisal may be to utilize fMRI paradigms like the affective Stroop task to probe cognition-emotion interactions . If mindWhen individuals are unable to marshal effective problem-solving to resolve a stressor, lack of a favorable resolution may lead to deployment of emotion regulation efforts to manage the emotional distress elicited by the stressful circumstance. Neuroimaging research has provided evidence for a reciprocal, dual-system neural network model of emotion regulation comprised of a dorsal brain system  subserving top-down cognitive control, and a ventral brain system  subserving bottom-up emotional impulses –135. TopImportantly, MBIs provide training in cultivating a state of mindful awareness and acceptance of the extant emotional response as a precondition for emotion regulation. While acceptance of aversive mental experience may itself result in reduced negative affect , mindfulSpeculatively, this “mindful reappraisal” process may involve spreading activation in a number of brain networks. Generating the state of mindfulness in the midst of a negative affective state may activate the ACC and dlPFC , 149, whThe effects of mindfulness-centered regulation of negative emotion might be measured with a standard emotion regulation paradigm [c.f. ], in whiIn addition to pro-regulatory effects on emotion, mindfulness training may facilitate neurocognitive regulation of the effects of stress on the autonomic nervous system. As addicts in treatment develop dispositional mindfulness through mindfulness training, they may be more able to engage prefrontal cortical modulation of the sympathetic “fight-or-flight” response via parasympathetic nervous system activation of the “vagal brake,” resulting in increased HRV and heart-rate deceleration in the face of stress or addictive cues , 154. ThThis hypothesis is consistent with evidence of the effects of mindfulness on neural function in dlPFC and ACC , 157, kein vivo, imaginally, or images of alcohol or drugs), and finally asked to use mindfulness skills to downregulate the resultant state of autonomic arousal.The effects of MBIs on cognitive regulation of autonomic cue-reactivity might be measured with a stress-primed cue-reactivity paradigm, in which participants are first exposed to a laboratory stress induction  is a taIndividuals in early recovery from addiction often attempt to suppress craving for drugs and alcohol as a means of maintaining abstinence. However, these suppression attempts often backfire, resulting in depletion of self-control resources , 179 andIn support of this hypothesis, changes in thought suppression have been shown to partially mediate effects of mindfulness training on alcohol use and drinking consequences . Furtherad libitum smoking session) in which skin conductance, heart rate, and craving responses could be measured throughout. If mindfulness enhances cognitive regulation of extinction learning, cue-elicited skin conductance, heart rate, and self-reported craving would be reduced following mindfulness training relative to an active control intervention, as would drug-use following the cue-exposure session.The effects of MBIs on cognitive regulation of extinction learning might be measured by combining neuroimaging, self-reported craving, and self-reported emotion regulatory strategy during a drug cue-reactivity paradigm. Pre- and post-mindfulness training, addicts could participate in cue-exposure sessions , this encounter may activate cortico-limbic-striatal circuits subserving drug-use action schemas. After completing a course in mindfulness training, the addict may become more aware of the automatic addictive habit as it is activated, allowing for top-down regulation of the precipitating negative emotional state and the bottom-up appetitive urge. Specifically, the individual may engage in mindful breathing to first disengage from and then restructure negative cognitive appraisals, thereby reducing limbic  activity, autonomic reactivity, and dysphoric emotions related to the stressor. Concurrently, the individual may become aware of when his attention has been automatically captured by the sight of people drinking in the window of the bar, and, through formal mindfulness practice, activate fronto-parietal mediated attentional networks to disengage and shift focus onto the neutral sensation of respiration. During this process, as sensations of craving arise, the individual may engage in metacognitive monitoring of these sensations, and in so doing, facilitate prefrontal down-regulation of limbic-striatal activation. As mindfulness of craving is sustained over time without drug-use, the sensations of craving may abate, promoting extinction learning to weaken associative linkages between conditioned addiction-related stimuli and the attendant conditioned appetitive response. Once working memory has been cleared of active representations of substance use, the individual may shift attention to savor non-drug related rewards, such as the sense of accomplishment that may arise from successfully resisting the temptation to drink , appreciating the beauty of the sunset on the walk home without being clouded by inebriation, or the comforting touch of a loved one upon returning home safe and sober. Through repeated practice of regulating addictive responses and extracting pleasure from life in the absence of substance use, the individual may re-establish healthy dopaminergic tone and foster neuroplasticity in brain areas subserving increased dispositional mindfulness.Ultimately, mindfulness may facilitate a novel, adaptive response to the canonical “people, places, and things” that tend to elicit addictive behavior as a scripted, habitual reaction. In so doing, the practice of mindfulness may attenuate stress reactivity and suppression while disrupting addictive automaticity, resulting in an increased ability to regulate and recover from addictive urges. The neurocognitive framework we have presented is intended to stimulate future research and facilitate the optimization of MBIs for the treatment of addiction. The tools of modern science have only begun to elucidate the many ways in which mindfulness training targets the risk chain of addiction at the attention-appraisal-emotion interface.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.