Musical Expertise Shapes Functional and Structural Brain Networks Independent of Absolute Pitch Ability.

Professional musicians are a popular model for investigating experience-dependent plasticity in human large-scale brain networks. A minority of musicians possess absolute pitch, the ability to name a tone without reference. The study of absolute pitch musicians provides insights into how a very specific talent is reflected in brain networks. Previous studies of the effects of musicianship and absolute pitch on large-scale brain networks have yielded highly heterogeneous findings regarding the localization and direction of the effects. This heterogeneity was likely influenced by small samples and vastly different methodological approaches. Here, we conducted a comprehensive multimodal assessment of effects of musicianship and absolute pitch on intrinsic functional and structural connectivity using a variety of commonly used and state-of-the-art multivariate methods in the largest sample to date (n = 153 female and male human participants; 52 absolute pitch musicians, 51 non-absolute pitch musicians, and 50 non-musicians). Our results show robust effects of musicianship in interhemispheric and intrahemispheric connectivity in both structural and functional networks. Crucially, most of the effects were replicable in both musicians with and without absolute pitch compared with non-musicians. However, we did not find evidence for an effect of absolute pitch on intrinsic functional or structural connectivity in our data: The two musician groups showed strikingly similar networks across all analyses. Our results suggest that long-term musical training is associated with robust changes in large-scale brain networks. The effects of absolute pitch on neural networks might be subtle, requiring very large samples or task-based experiments to be detected.SIGNIFICANCE STATEMENT A question that has fascinated neuroscientists, psychologists, and musicologists for a long time is how musicianship and absolute pitch, the rare talent to name a tone without reference, are reflected in large-scale networks of the human brain. Much is still unknown as previous studies have reported widely inconsistent results based on small samples. Here, we investigate the largest sample of musicians and non-musicians to date (n = 153) using a multitude of established and novel analysis methods. Results provide evidence for robust effects of musicianship on functional and structural networks that were replicable in two separate groups of musicians and independent of absolute pitch ability.

Altered limbic and autonomic processing supports brain-heart axis in Takotsubo syndrome.

AIMS: Takotsubo syndrome (TTS) is characterized by acute left ventricular dysfunction often triggered by emotional or physical stress. Severe activation of the sympathetic nervous system with catecholamine release caused by a dysfunctional limbic system has been proposed as a potential mechanism. We hypothesize that brain regions responsible for autonomic integration and/or limbic processing might be involved in the development of TTS. Here, we investigated alterations in resting state functional connectivity in TTS patients compared with healthy controls. METHODS AND RESULTS: Using brain functional magnetic resonance imaging (fMRI), resting state functional connectivity has been assessed in 15 subjects with TTS and 39 healthy controls. Network-based statistical analyses were conducted to identify subnetworks with altered resting state functional connectivity. Sympathetic and parasympathetic networks have been constructed in addition to the default mode network and whole-brain network. We found parasympathetic- and sympathetic-associated subnetworks both showing reduced resting state functional connectivity in TTS patients compared with controls. Important brain regions constituting parasympathetic- and sympathetic-associated subnetworks included the amygdala, hippocampus, and insula as well as cingulate, parietal, temporal, and cerebellar regions. Additionally, the default mode network as well as limbic regions in the whole-brain analysis demonstrated reduced resting state functional connectivity in TTS, including the hippocampus, parahippocampal, and medial prefrontal regions. CONCLUSION: For the first time, we demonstrate hypoconnectivity of central brain regions associated with autonomic functions and regulation of the limbic system in patients with TTS. These findings suggest that autonomic-limbic integration might play an important role in the pathophysiology and contribute to the understanding of TTS.

Expertise-related functional brain network efficiency in healthy older adults.

BACKGROUND: In view of age-related brain changes, identifying factors that are associated with healthy aging are of great interest. In the present study, we compared the functional brain network characteristics of three groups of healthy older participants aged 61-75 years who had a different cognitive and motor training history (multi-domain group: participants who had participated in a multi-domain training; visuomotor group: participants who had participated in a visuomotor training; control group: participants with no specific training history). The study's basic idea was to examine whether these different training histories are associated with differences in behavioral performance as well as with task-related functional brain network characteristics. Based on a high-density electroencephalographic measurement one year after training, we calculated graph-theoretical measures representing the efficiency of functional brain networks. RESULTS: Behaviorally, the multi-domain group performed significantly better than the visuomotor and the control groups on a multi-domain task including an inhibition domain, a visuomotor domain, and a spatial navigation domain. In terms of the functional brain network features, the multi-domain group showed significantly higher functional connectivity in a network encompassing visual, motor, executive, and memory-associated brain areas in the theta frequency band compared to the visuomotor group. These brain areas corresponded to the multi-domain task demands. Furthermore, mean connectivity of this network correlated positively with performance across both the multi-domain and the visuomotor group. In addition, the multi-domain group showed significantly enhanced processing efficiency reflected by a higher mean weighted node degree (strength) of the network as compared to the visuomotor group. CONCLUSIONS: Taken together, our study shows expertise-dependent differences in task-related functional brain networks. These network differences were evident even a year after the acquisition of the different expertise levels. Hence, the current findings can foster understanding of how expertise is positively associated with brain functioning during aging.

The "silent" imprint of musical training.

Playing a musical instrument at a professional level is a complex multimodal task requiring information integration between different brain regions supporting auditory, somatosensory, motor, and cognitive functions. These kinds of task-specific activations are known to have a profound influence on both the functional and structural architecture of the human brain. However, until now, it is widely unknown whether this specific imprint of musical practice can still be detected during rest when no musical instrument is used. Therefore, we applied high-density electroencephalography and evaluated whole-brain functional connectivity as well as small-world topologies (i.e., node degree) during resting state in a sample of 15 professional musicians and 15 nonmusicians. As expected, musicians demonstrate increased intra- and interhemispheric functional connectivity between those brain regions that are typically involved in music perception and production, such as the auditory, the sensorimotor, and prefrontal cortex as well as Broca's area. In addition, mean connectivity within this specific network was positively related to musical skill and the total number of training hours. Thus, we conclude that musical training distinctively shapes intrinsic functional network characteristics in such a manner that its signature can still be detected during a task-free condition. Hum Brain Mapp 37:536-546, 2016. © 2015 Wiley Periodicals, Inc.

The Influence of Pre-stimulus EEG Activity on Reaction Time During a Verbal Sternberg Task is Related to Musical Expertise.

Previous work highlighted the possibility that musical training has an influence on cognitive functioning. The suggested reason for this influence is the strong recruitment of attention, planning, and working memory functions during playing a musical instrument. The purpose of the present work was twofold, namely to evaluate the general relationship between pre-stimulus electrophysiological activity and cognition, and more specifically the influence of musical expertise on working memory functions. With this purpose in mind, we used covariance mapping analyses to evaluate whether pre-stimulus electroencephalographic activity is predictive for reaction time during a visual working memory task (Sternberg paradigm) in musicians and non-musicians. In line with our hypothesis, we replicated previous findings pointing to a general predictive value of pre-stimulus activity for working memory performance. Most importantly, we also provide first evidence for an influence of musical expertise on working memory performance that could distinctively be predicted by pre-stimulus spectral power. Our results open novel perspectives for better comprehending the vast influences of musical expertise on cognition.

MRI with and without a high-density EEG cap--what makes the difference?

Besides the benefit of combining electroencephalography (EEG) and magnetic resonance imaging (MRI), much effort has been spent to develop algorithms aimed at successfully cleaning the EEG data from MRI-related gradient and ballistocardiological artifacts. However, there are also studies showing a negative influence of the EEG on MRI data quality. Therefore, in the present study, we focused for the first time on the influence of the EEG on morphometric measurements of T1-weighted MRI data (voxel- and surfaced-based morphometry). Here, we demonstrate a strong influence of the EEG on cortical thickness, surface area, and volume as well as subcortical volumes due to local EEG-related inhomogeneities of the static magnetic (B0) and the gradient field (B1). In a second step, we analyzed the signal-to-noise ratios for both the anatomical and the functional data when recorded simultaneously with EEG and MRI and compared them to the ratios of the MRI data without simultaneous EEG measurements. These analyses revealed consistently lower signal-to-noise ratios for anatomical as well as functional MRI data during simultaneous EEG registration. In contrast, further analyses of T2*-weighted images provided reliable results independent of whether including the individuals' T1-weighted image with or without the EEG cap in the fMRI preprocessing stream. Based on our findings, we strongly recommend against using the structural images obtained during simultaneous EEG-MRI recordings for further anatomical data analysis.

Music and language expertise influence the categorization of speech and musical sounds: behavioral and electrophysiological measurements.

In this study, we used high-density EEG to evaluate whether speech and music expertise has an influence on the categorization of expertise-related and unrelated sounds. With this purpose in mind, we compared the categorization of speech, music, and neutral sounds between professional musicians, simultaneous interpreters (SIs), and controls in response to morphed speech-noise, music-noise, and speech-music continua. Our hypothesis was that music and language expertise will strengthen the memory representations of prototypical sounds, which act as a perceptual magnet for morphed variants. This means that the prototype would "attract" variants. This so-called magnet effect should be manifested by an increased assignment of morphed items to the trained category, by a reduced maximal slope of the psychometric function, as well as by differential event-related brain responses reflecting memory comparison processes (i.e., N400 and P600 responses). As a main result, we provide first evidence for a domain-specific behavioral bias of musicians and SIs toward the trained categories, namely music and speech. In addition, SIs showed a bias toward musical items, indicating that interpreting training has a generic influence on the cognitive representation of spectrotemporal signals with similar acoustic properties to speech sounds. Notably, EEG measurements revealed clear distinct N400 and P600 responses to both prototypical and ambiguous items between the three groups at anterior, central, and posterior scalp sites. These differential N400 and P600 responses represent synchronous activity occurring across widely distributed brain networks, and indicate a dynamical recruitment of memory processes that vary as a function of training and expertise.

LPS-induced cytokine production in human monocytes and macrophages.

Lipopolysaccharide (LPS) from Gram-negative bacteria is one of the most potent innate immune-activating stimuli known. Here we review the current understanding of LPS effects on human monocyte and macrophage function. We provide an overview of LPS signal transduction with attention given to receptor cooperativity and species differences in LPS responses, as well as the role of tyrosine phosphorylation and lysine acetylation in signalling. We also review LPS-regulated transcription, with emphasis on chromatin remodeling and primary versus secondary transcriptional control mechanisms. Finally, we review the regulation and function of LPS-inducible cytokines produced by human monocytes and macrophages including TNFα, the IL-1 family, IL-6, IL-8, the IL-10 family, the IL-12 family, IL-15 and TGFß.

Functional connectivity changes in the delta frequency band following trauma treatment in complex trauma and dissociative disorder patients.

Objective: Phase-oriented trauma treatment is efficacious in the treatment of complex trauma and dissociative disorder patients. However, the neural correlates of this therapeutic effect are not yet well-understood. In the current study we investigated whether patients show a strengthening in functional network connectivity in the delta frequency band (1-3.5 Hz) over the course of phase-oriented inpatient trauma treatment while they performed an emotion regulation task. Further, we examined whether neural changes were associated with symptom reduction and improvement in emotion regulation skills. Methods: Before and after 8 weeks of treatment, electroencephalography (EEG) was acquired in patients (n = 28) with a complex posttraumatic stress disorder (cPTSD) or complex dissociative disorder (CDD). They also completed clinical and emotion regulation questionnaires. To delimit data variability, patients participated as one dissociative part that is referred to as Apparently Normal Part (ANP). Patients' data were compared to a matched healthy control croup (n = 38), also measured twice. Results: Prior to treatment, functional connectivity was significantly lower in patients compared to controls during cognitive reappraisal of unpleasant pictures and passive viewing of unpleasant and neutral pictures. These hypoconnected networks largely overlapped with networks typically activated during the recall of (emotional) autobiographical memories. Functional connectivity strength within these networks significantly increased following treatment and was comparable to controls. Patients showed symptom reduction across various clinical domains and improvement in the use of cognitive reappraisal as emotion regulation strategy. Treatment-related network normalizations were not related to changes in questionnaire data. Conclusion: Phase-oriented treatment may strengthen connections between regions that are activated during autobiographical recall. These findings encourage further investigation of this circuitry as a therapeutic target in cPTSD and CDD patients. Clinial trial registration: www.ClinicalTrials.gov, identifier: NCT02459340, https://www.kofam.ch/de/studienportal/suche/149284/studie/26681.

Resting-state functional connectivity in patients with a complex PTSD or complex dissociative disorder before and after inpatient trauma treatment.

INTRODUCTION: Recent research suggests that traumatized patients are characterized by disrupted resting-state functional connectivity. We examined whether neural networks involved in resting-state change over the course of a phase-oriented inpatient treatment for complex traumatized and dissociative disorder patients. We also investigated associations between these network alterations and clinical symptoms and emotion regulation skills. METHODS: Pre- and post-treatment, electroencephalography (EEG) was recorded during resting-state in patients (n = 23) with a complex dissociative disorder (CDD) or complex posttraumatic stress disorder (cPTSD). Patients also completed clinical and emotion regulation questionnaires. To reduce variance in the collected data, patients were exclusively tested as one prototypical dissociative part referred to as Apparently Normal Part (ANP). Functional network connectivity was examined and compared with a matched healthy control group (n = 37), also measured twice. RESULTS: Prior to treatment and compared with controls, patients had a significantly lower functional connectivity strength within eyes-open and eyes-closed resting-state networks in the theta and alpha frequency band. Following treatment, functional connectivity strength within these networks was comparable to the control group and comprised areas belonging to the default mode network (DMN) and prefrontal as well as anterior cingulate control regions. Treatment-related network normalizations in the theta frequency band were associated with a self-reported increase in the use of cognitive reappraisal strategies and reduction in emotion regulation difficulties. CONCLUSION: Phase-oriented trauma treatment can strengthen resting-state network connectivity and can increase the capacity of complex traumatized and dissociative patients as ANP to handle emotional challenges effectively.

Heterogeneity of EEG resting-state brain networks in absolute pitch.

The neural basis of absolute pitch (AP), the ability to effortlessly identify a musical tone without an external reference, is poorly understood. One of the key questions is whether perceptual or cognitive processes underlie the phenomenon, as both sensory and higher-order brain regions have been associated with AP. To integrate the perceptual and cognitive views on AP, here, we investigated joint contributions of sensory and higher-order brain regions to AP resting-state networks. We performed a comprehensive functional network analysis of source-level EEG in a large sample of AP musicians (n = 54) and non-AP musicians (n = 51), adopting two analysis approaches: First, we applied an ROI-based analysis to examine the connectivity between the auditory cortex and the dorsolateral prefrontal cortex (DLPFC) using several established functional connectivity measures. This analysis is a replication of a previous study which reported increased connectivity between these two regions in AP musicians. Second, we performed a whole-brain network-based analysis on the same functional connectivity measures to gain a more complete picture of the brain regions involved in a possibly large-scale network supporting AP ability. In our sample, the ROI-based analysis did not provide evidence for an AP-specific connectivity increase between the auditory cortex and the DLPFC. The whole-brain analysis revealed three networks with increased connectivity in AP musicians comprising nodes in frontal, temporal, subcortical, and occipital areas. Commonalities of the networks were found in both sensory and higher-order brain regions of the perisylvian area. Further research will be needed to confirm these exploratory results.

Takotsubo syndrome: How the broken heart deals with negative emotions.

OBJECTIVES: Patients suffering from Takotsubo syndrome have a higher prevalence of anxiety and depressive disorders compared to those with acute myocardial infarction and might thus show impaired regulation and processing of emotions. METHODS: In this cross-sectional study, neural activity during an emotional picture processing task was examined in 26 Takotsubo patients (on average 27 months after the Takotsubo event) and 22 healthy age- and gender-matched control subjects undergoing functional magnetic resonance imaging. Imaging data were analyzed with two complementary approaches: First, univariate analysis was used to detect brain regions showing condition-specific differences in mean neural activity between groups. Second, multivariate pattern analysis was applied to decode the experimental conditions from individual activity patterns. RESULTS: In the univariate analysis approach, patients showed lower bilateral superior parietal activity during the processing of negative expected pictures compared to the control subjects. The multivariate pattern analysis revealed group differences in decoding negative versus neutral pictures from a widespread network consisting of frontal, parietal, occipital, and cerebellar brain regions. Additionally, differences in decoding the expectation of a negative versus positive upcoming picture were observed in the visual cortex. CONCLUSION: The lower involvement of brain regions observed in Takotsubo patients suggests an impairment in emotion regulation, which might be of etiological importance in this brain-heart disease.

Functional reorganization of neural networks involved in emotion regulation following trauma therapy for complex trauma disorders.

OBJECTIVES: We investigated whether patients with complex interpersonal trauma engage neural networks that are commonly activated during cognitive reappraisal and responding naturally to affect-laden images. In this naturalistic study, we examined whether trauma treatment not only reduces symptoms but also changes neural networks involved in emotional control. METHODS: Before and after eight weeks of phase-oriented inpatient trauma treatment, patients (n = 28) with complex posttraumatic stress disorder (cPTSD) and complex dissociative disorders (CDD) performed a cognitive reappraisal task while electroencephalography (EEG) was registered. Patients were measured as a prototypical dissociative part that aims to fulfill daily life goals while avoiding traumatic memories and associated dissociative parts. Matched healthy controls (n = 38) were measured twice as well. We examined task-related functional connectivity and assessed self-reports of clinical symptoms and emotion regulation skills. RESULTS: Prior to treatment and compared to controls, patients showed hypoconnectivity within neural networks involved in emotional downregulation while reappraising affect-eliciting pictures as well as viewing neutral and affect-eliciting pictures. Following treatment, connectivity became normalized in these networks comprising regions associated with cognitive control and memory. Additionally, patients showed a treatment-related reduction of negative but not of positive dissociative symptoms. CONCLUSIONS: This is the first study demonstrating that trauma-focused treatment was associated with favorable changes in neural networks involved in emotional control. Emotional overregulation manifesting as negative dissociative symptoms was reduced but not emotional underregulation, manifesting as positive dissociative symptoms.

The interpreter's brain during rest - Hyperconnectivity in the frontal lobe.

Language in its highest complexity is a unique human faculty with simultaneous translation being among the most demanding language task involving both linguistic and executive functions. In this context, bilingually grown up individuals as well as simultaneous interpreters (SIs) represent appropriate groups for studying expertise-related neural adaptations in the human brain. The present study was performed to examine if a domain-specific neural network activation pattern, constituted by brain regions involved in speech processing as well as cognitive control mechanisms can be detected during a task-free resting state condition. To investigate this, electroencephalographic (EEG) data were recorded from 16 SIs and 16 age and gender-matched multilingual control subjects. Graph-theoretical network analyses revealed interhemispheric hyperconnectivity between the ventral part of the prefrontal cortex (pars opercularis and pars triangularis) and the dorsolateral prefrontal cortex (DLPFC) in language experts compared to multilingual controls in the alpha frequency range. This finding suggests that the high cognitive demands placed on simultaneous interpreting lead to an increased neural communication between prefrontal brain regions essentially engaged in supporting executive control-a neural fingerprint that is even detectable during rest.

Takotsubo Syndrome - Predictable from brain imaging data.

Takotsubo syndrome (TTS) is characterized by acute left ventricular dysfunction, with a hospital-mortality rate similar to acute coronary syndrome (ACS). However, the aetiology of TTS is still unknown. In the present study, a multivariate pattern analysis using machine learning with multimodal magnetic resonance imaging (MRI) data of the human brain of TTS patients and age- and gender-matched healthy control subjects was performed. We found consistent structural and functional alterations in TTS patients compared to the control group. In particular, anatomical and neurophysiological measures from brain regions constituting the emotional-autonomic control system contributed to a prediction accuracy of more than 82%. Thus, our findings demonstrate homogeneous neuronal alterations in TTS patients and substantiate the importance of the concept of a brain-heart interaction in TTS.

Inhibition of nicotinamide phosphoribosyltransferase modifies LPS-induced inflammatory responses of human monocytes.

Recent studies have identified enzymes that use NAD as a substrate, thus contributing to its net consumption. To maintain the intracellular pool, NAD is re-synthesized by a salvage pathway using nicotinamide, the by-product generated by the enzymatic cleavage of NAD. Enzymes involved in NAD re-synthesis include nicotinamide phosphoribosyltransferase (NAMPT) and nicotinamide mononucleotide adenylyltransferase. Our studies show, that NAMPT was substantially up-regulated by LPS in primary human monocytes, suggesting that it may be especially required during the process of monocyte activation. To evaluate the contribution of the NAD rescue pathway to LPS-induced biological responses in human monocytes, we used APO866, a well-characterized inhibitor of NAMPT. Concomitant with the inhibition of NAMPT, LPS-induced TNF-α protein synthesis declined, while TNF-α mRNA levels were minimally affected. Moreover, APO866 strongly decreased the production of reactive oxygen species (ROS), increased surface expression of the NAD-consuming enzyme CD38, and modified the production of selective eicosanoids. We further demonstrate that protein ADP-ribosylation was strongly reduced, indicating a possible link between this post-translational protein modification and human monocyte inflammatory responses. Despite a substantial reduction in intracellular NAD levels, activated monocytes were resistant to apoptosis, while resting monocytes were not. Taken together, our data suggest that activated monocytes strongly depend on the NAD salvage pathway to mount an appropriate inflammatory response. Their survival is not affected by NAD-depletion, probably as a result of LPS-mediated anti-apoptotic signals.

The BDNF Val66Met polymorphism modulates sleep intensity: EEG frequency- and state-specificity.

STUDY OBJECTIVES: EEG slow waves are the hallmark of deep NREM sleep and may reflect the restorative functions of sleep. Evidence suggests that increased sleep slow waves after sleep deprivation reflect plastic synaptic processes, and that brain-derived neurotrophic factor (BDNF) is causally involved in their homeostatic regulation. The functional Val66Met polymorphism of the gene encoding pro-BDNF causes impaired activity-dependent secretion of mature BDNF protein. We investigated whether this polymorphism contributes to the pronounced inter-individual variation in sleep slow wave activity (SWA) in humans. SETTING: Sleep laboratory in temporal isolation unit. PARTICIPANTS: Eleven heterozygous Met allele carriers and 11 individually sex- and age-matched Val/Val homozygotes. INTERVENTIONS: Forty hours prolonged wakefulness. MEASUREMENTS AND RESULTS: Cognitive performance, subjective state, and waking and sleep EEG in baseline and after sleep deprivation were studied. Val/Val homozygotes showed better response accuracy than Met allele carriers on a verbal 2-back working memory task. This difference did not reflect genotype-dependent differences in sleepiness, well-being, or sustained attention. In baseline and recovery nights, deep stage 4 sleep and NREM sleep intensity as quantified by EEG SWA (0.75-4.5 Hz) were higher in Val/Val compared to Val/Met genotype. Similar to sleep deprivation, the difference was most pronounced in the first NREM sleep episode. By contrast, increased activity in higher EEG frequencies (> 6 Hz) in wakefulness and REM sleep was distinct from the effects of prolonged wakefulness. CONCLUSION: BDNF contributes to the regulation of sleep slow wave oscillations, suggesting that genetically determined variation in neuronal plasticity modulates NREM sleep intensity in humans.

Review: NAD +: a modulator of immune functions.

Latterly, nicotinamide adenine dinucleotide (NAD+) has emerged as a molecule with versatile functions and of enormous impact on the maintenance of cell integrity. Besides playing key roles in almost all major aspects of energy metabolism, there is mounting evidence that NAD+ and its degradation products affect various biological activities including calcium homeostasis, gene transcription, DNA repair, and intercellular communication. This review is aimed at giving a brief insight into the life cycle of NAD+ in the cell, referring to synthesis, action and degradation aspects. With respect to their immunological relevance, the importance and function of the major NAD+ metabolizing enzymes, namely CD38/CD157, ADP-ribosyltransferases (ARTs), poly-ADP-ribose-polymerases (PARPs), and sirtuins are summarized and roles of NAD+ and its main degradation product adenosine 5'-diphosphoribose (ADPR) in cell signaling are discussed. In addition, an outline of the variety of immunological processes depending on the activity of nicotinamide phosphoribosyltransferase (Nampt), the key enzyme of the salvage pathway of NAD+ synthesis, is presented. Taken together, an efficient supply of NAD+ seems to be a crucial need for a multitude of cell functions, underlining the yet only partly revealed potency of this small molecule to influence cell fate.

Involvement of P2X receptors in the NAD(+)-induced rise in [Ca (2+)] (i) in human monocytes.

In the present study, we show that the extracellular addition of nicotinamide adenine dinucleotide (NAD(+)) induces a transient rise in [Ca(2+)](i) in human monocytes caused by an influx of extracellular calcium. The NAD(+)-induced Ca(2+) response was prevented by adenosine triphosphate (ATP), suggesting the involvement of ATP receptors. Of the two subtypes of ATP receptors (P2X and P2Y), the P2X receptors were considered the most likely candidates. By the use of subtype preferential agonists and antagonists, we identified P2X(1), P2X(4), and P2X(7) receptors being engaged in the NAD(+)-induced rise in [Ca(2+)](i). Among the P2X receptor subtypes, the P2X(7) receptor is unique in facilitating the induction of nonselective pores that allow entry of ethidium upon stimulation with ATP. In monocytes, opening of P2X(7) receptor-dependent pores strongly depends on specific ionic conditions. Measuring pore formation in response to NAD(+), we found that NAD(+) unlike ATP lacks the ability to induce this pore-forming response. Whereas as little as 100 muM ATP was sufficient to activate the nonselective pore, NAD(+) at concentrations up to 2 mM had no effect. Taken together, these data indicate that despite similarities in the action of extracellular NAD(+) and ATP there are nucleotide-specific variations. So far, common and distinct features of the two nucleotides are only beginning to be understood.