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The nervous system uses fast- and slow-adapting sensory detectors in parallel to enable neuronal representations of external states and their temporal dynamics. It is unknown whether this dichotomy also applies to internal representations that have no direct correlation in the physical world. Here we find that two distinct dopamine (DA) neuron subtypes encode either a state or its rate-of-change. In mice performing a reward-seeking task, we found that the animal's behavioral state and rate-of-change were encoded by the sustained activity of DA neurons in medial ventral tegmental area (VTA) DA neurons and transient activity in lateral VTA DA neurons, respectively. The neural activity patterns of VTA DA cell bodies matched DA release patterns within anatomically defined mesoaccumbal pathways. Based on these results, we propose a model in which the DA system uses two parallel lines for proportional-differential encoding of a state variable and its temporal dynamics., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The sucrose preference test (SPT) measures the relative preference of sucrose over water to assess hedonic behaviors in rodents. Yet, it remains uncertain to what extent the SPT reflects other behavioral components, such as learning, memory, motivation, and choice. Here, we conducted an experimental and computational decomposition of mouse behavior in the SPT and discovered previously unrecognized behavioral subcomponents associated with changes in sucrose preference. We show that acute and chronic stress have sex-dependent effects on sucrose preference, but anhedonia was observed only in response to chronic stress in male mice. Additionally, reduced sucrose preference induced by optogenetics is not always indicative of anhedonia but can also reflect learning deficits. Even small variations in experimental conditions influence behavior, task outcome and interpretation. Thus, an ostensibly simple behavioral task can entail high levels of complexity, demonstrating the need for careful dissection of behavior into its subcomponents when studying the underlying neurobiology., (© 2023. The Author(s).)

We collected data on mass mortality in Sandwich terns (Thalasseus sandvicensis) during the 2022 breeding season in the Netherlands. Mortality was associated with at least 2 variants of highly pathogenic avian influenza A(H5N1) virus clade 2.3.4.4b. We report on carcass removal efforts relative to survival in colonies. Mitigation strategies urgently require structured research.

Nicotine stimulates the dopamine (DA) system, which is essential for its rewarding effect. Nicotine is also aversive at high doses; yet, our knowledge about nicotine's dose-dependent effects on DA circuits remains limited. Here, we demonstrate that high doses of nicotine, which induce aversion-related behavior in mice, cause biphasic inhibitory and excitatory responses in VTA DA neurons that can be dissociated by distinct projections to lateral and medial nucleus accumben subregions, respectively. Guided by computational modeling, we performed a pharmacological investigation to establish that inhibitory effects of aversive nicotine involve desensitization of α4β2 and activation of α7 nicotinic acetylcholine receptors. We identify α7-dependent activation of upstream GABA neurons in the laterodorsal tegmentum (LDT) as a key regulator of heterogeneous DA release following aversive nicotine. Finally, inhibition of LDT GABA terminals in VTA prevents nicotine aversion. Together, our findings provide a mechanistic circuit-level understanding of nicotine's dose-dependent effects on reward and aversion., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Ventral tegmental area (VTA) dopamine (DA) neurons are often thought to uniformly encode reward prediction errors. Conversely, DA release in the nucleus accumbens (NAc), the prominent projection target of these neurons, has been implicated in reinforcement learning, motivation, aversion, and incentive salience. This contrast between heterogeneous functions of DA release versus a homogeneous role for DA neuron activity raises numerous questions regarding how VTA DA activity translates into NAc DA release. Further complicating this issue is increasing evidence that distinct VTA DA projections into defined NAc subregions mediate diverse behavioral functions. Here, we evaluate evidence for heterogeneity within the mesoaccumbal DA system and argue that frameworks of DA function must incorporate the precise topographic organization of VTA DA neurons to clarify their contribution to health and disease.

Pain decreases the activity of many ventral tegmental area (VTA) dopamine (DA) neurons, yet the underlying neural circuitry connecting nociception and the DA system is not understood. Here we show that a subpopulation of lateral parabrachial (LPB) neurons is critical for relaying nociceptive signals from the spinal cord to the substantia nigra pars reticulata (SNR). SNR-projecting LPB neurons are activated by noxious stimuli and silencing them blocks pain responses in two different models of pain. LPB-targeted and nociception-recipient SNR neurons regulate VTA DA activity directly through feed-forward inhibition and indirectly by inhibiting a distinct subpopulation of VTA-projecting LPB neurons thereby reducing excitatory drive onto VTA DA neurons. Correspondingly, ablation of SNR-projecting LPB neurons is sufficient to reduce pain-mediated inhibition of DA release in vivo. The identification of a neural circuit conveying nociceptive input to DA neurons is critical to our understanding of how pain influences learning and behavior., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The excitatory synapse between hippocampal CA3 and CA1 pyramidal neurons exhibits long-term potentiation (LTP), a positive feedback process implicated in learning and memory in which postsynaptic depolarization strengthens synapses, promoting further depolarization. Without mechanisms for interrupting positive feedback, excitatory synapses could strengthen inexorably, corrupting memory storage. Here, we reveal a hidden form of inhibitory synaptic plasticity that prevents accumulation of excitatory LTP. We developed a knockin mouse that allows optical control of endogenous α5-subunit-containing γ-aminobutyric acid (GABA) A receptors (α5-GABARs). Induction of excitatory LTP relocates α5-GABARs, which are ordinarily extrasynaptic, to inhibitory synapses, quashing further NMDA receptor activation necessary for inducing more excitatory LTP. Blockade of α5-GABARs accelerates reversal learning, a behavioral test for cognitive flexibility dependent on repeated LTP. Hence, inhibitory synaptic plasticity occurs in parallel with excitatory synaptic plasticity, with the ensuing interruption of the positive feedback cycle of LTP serving as a possible critical early step in preserving memory., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

In this issue of Neuron, Corkrum et al. (2020) demonstrate an unexpected role for dopamine D 1 receptors on astrocytes located in the nucleus accumbens, a key structure of the brain's reward system. Activation of these receptors mediates dopamine-evoked depression of excitatory synaptic transmission, which contributes to amphetamine's psychomotor effects., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Chronic stress (CS) is a major risk factor for the development of depression. Here, we demonstrate that CS-induced hyperactivity in ventral tegmental area (VTA)-projecting lateral habenula (LHb) neurons is associated with increased passive coping (PC), but not anxiety or anhedonia. LHb→VTA neurons in mice with increased PC show increased burst and tonic firing as well as synaptic adaptations in excitatory inputs from the entopeduncular nucleus (EP). In vivo manipulations of EP→LHb or LHb→VTA neurons selectively alter PC and effort-related motivation. Conversely, dorsal raphe (DR)-projecting LHb neurons do not show CS-induced hyperactivity and are targeted indirectly by the EP. Using single-cell transcriptomics, we reveal a set of genes that can collectively serve as biomarkers to identify mice with increased PC and differentiate LHb→VTA from LHb→DR neurons. Together, we provide a set of biological markers at the level of genes, synapses, cells, and circuits that define a distinctive CS-induced behavioral phenotype., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The dorsal raphe (DR) is a heterogeneous nucleus containing dopamine (DA), serotonin (5HT), γ-aminobutyric acid (GABA) and glutamate neurons. Consequently, investigations of DR circuitry require Cre-driver lines that restrict transgene expression to precisely defined cell populations. Here, we present a systematic evaluation of mouse lines targeting neuromodulatory cells in the DR. We find substantial differences in specificity between lines targeting DA neurons, and in penetrance between lines targeting 5HT neurons. Using these tools to map DR circuits, we show that populations of neurochemically distinct DR neurons are arranged in a stereotyped topographical pattern, send divergent projections to amygdala subnuclei, and differ in their presynaptic inputs. Importantly, targeting DR DA neurons using different mouse lines yielded both structural and functional differences in the neural circuits accessed. These results provide a refined model of DR organization and support a comparative, case-by-case evaluation of the suitability of transgenic tools for any experimental application.

Ventral tegmental area (VTA) dopamine (DA) neurons play a central role in mediating motivated behaviors, but the circuitry through which they signal positive and negative motivational stimuli is incompletely understood. Using in vivo fiber photometry, we simultaneously recorded activity in DA terminals in different nucleus accumbens (NAc) subnuclei during an aversive and reward conditioning task. We find that DA terminals in the ventral NAc medial shell (vNAcMed) are excited by unexpected aversive outcomes and to cues that predict them, whereas DA terminals in other NAc subregions are persistently depressed. Excitation to reward-predictive cues dominated in the NAc lateral shell and was largely absent in the vNAcMed. Moreover, we demonstrate that glutamatergic (VGLUT2-expressing) neurons in the lateral hypothalamus represent a key afferent input for providing information about aversive outcomes to vNAcMed-projecting DA neurons. Collectively, we reveal the distinct functional contributions of separate mesolimbic DA subsystems and their afferent pathways underlying motivated behaviors. VIDEO ABSTRACT., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Hyperdopaminergic states in mental disorders are associated with disruptive deficits in decision making. However, the precise contribution of topographically distinct mesencephalic dopamine pathways to decision-making processes remains elusive. Here we show, using a multidisciplinary approach, how hyperactivity of ascending projections from the ventral tegmental area (VTA) contributes to impaired flexible decision making in rats. Activation of the VTA-nucleus accumbens pathway leads to insensitivity to loss and punishment due to impaired processing of negative reward prediction errors. In contrast, activation of the VTA-prefrontal cortex pathway promotes risky decision making without affecting the ability to choose the economically most beneficial option. Together, these findings show how malfunction of ascending VTA projections affects value-based decision making, suggesting a potential mechanism through which increased forebrain dopamine signaling leads to aberrant behavior, as is seen in substance abuse, mania, and after dopamine replacement therapy in Parkinson's disease.

Mesolimbic dopamine (DA) neurons play a central role in motivation and reward processing. Although the activity of these mesolimbic DA neurons is controlled by afferent inputs, little is known about the circuits in which they are embedded. Using retrograde tracing, electrophysiology, optogenetics, and behavioral assays, we identify principles of afferent-specific control in the mesolimbic DA system. Neurons in the medial shell subdivision of the nucleus accumbens (NAc) exert direct inhibitory control over two separate populations of mesolimbic DA neurons by activating different GABA receptor subtypes. In contrast, NAc lateral shell neurons mainly synapse onto ventral tegmental area (VTA) GABA neurons, resulting in disinhibition of DA neurons that project back to the NAc lateral shell. Lastly, we establish a critical role for NAc subregion-specific input to the VTA underlying motivated behavior. Collectively, our results suggest a distinction in the incorporation of inhibitory inputs between different subtypes of mesolimbic DA neurons., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Motivational deficits are a key symptom in multiple psychiatric disorders, including major depressive disorder, schizophrenia and addiction. A likely neural substrate for these motivational deficits is the brain dopamine (DA) system. In particular, DA signalling in the nucleus accumbens, which originates from DA neurons in the ventral tegmental area (VTA), has been identified as a crucial substrate for effort-related and activational aspects of motivation. Unravelling how VTA DA neuronal activity relates to motivational behaviours is required to understand how motivational deficits in psychiatry can be specifically targeted. In this study, we therefore used designer receptors exclusively activated by designer drugs (DREADD) in TH:Cre rats, in order to determine the effects of chemogenetic DA neuron activation on different aspects of motivational behaviour. We found that chemogenetic activation of DA neurons in the VTA, but not substantia nigra, significantly increased responding for sucrose under a progressive ratio schedule of reinforcement. More specifically, high effort exertion was characterized by increased initiations of reward-seeking actions. This effect was dependent on effort requirements and instrumental contingencies, but was not affected by sucrose pre-feeding. Together, these findings indicate that VTA DA neuronal activation drives motivational behaviour by facilitating action initiation. With this study, we show that enhancing excitability of VTA DA neurons is a viable strategy to improve motivational behaviour., (Copyright © 2017 Elsevier B.V. and ECNP. All rights reserved.)

Linking neural circuit activation at whole-brain level to neuronal activity at cellular level remains one of the major challenges in neuroscience research. We set up a novel functional neuroimaging approach to map global effects of locally induced activation of specific midbrain projection neurons using chemogenetics (Designer Receptors Exclusively Activated by Designer Drugs (DREADD)-technology) combined with pharmacological magnetic resonance imaging (phMRI) in the rat mesocorticolimbic system. Chemogenetic activation of DREADD-targeted mesolimbic or mesocortical pathways, i.e. projections from the ventral tegmental area (VTA) to the nucleus accumbens (NAcc) or medial prefrontal cortex (mPFC), respectively, induced significant blood oxygenation level-dependent (BOLD) responses in areas with DREADD expression, but also in remote defined neural circuitry without DREADD expression. The time-course of brain activation corresponded with the behavioral output measure, i.e. locomotor (hyper)activity, in the mesolimbic pathway-targeted group. Chemogenetic activation specifically increased neuronal activity, whereas functional connectivity assessed with resting state functional MRI (rs-fMRI) remained stable. Positive and negative BOLD responses distinctively reflected simultaneous ventral pallidum activation and substantia nigra pars reticulata deactivation, respectively, demonstrating the concept of mesocorticolimbic network activity with concurrent activation of the direct and indirect pathways following stimulation of specific midbrain projection neurons. The presented methodology provides straightforward and widely applicable opportunities to elucidate relationships between local neuronal activity and global network activity in a controllable manner, which will increase our understanding of the functioning and dysfunctioning of large-scale neuronal networks in health and disease., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Dopamine (DA) signalling in the brain is necessary for feeding behaviour, and alterations in the DA system have been linked to obesity. However, the precise role of DA in the control of food intake remains debated. On the one hand, food reward and motivation are associated with enhanced DA activity. On the other hand, psychostimulant drugs that increase DA signalling suppress food intake. This poses the questions of how endogenous DA neuronal activity regulates feeding, and whether enhancing DA neuronal activity would either promote or reduce food intake., Methods: Here, we used designer receptors exclusively activated by designer drugs (DREADD) technology to determine the effects of enhancing DA neuronal activity on feeding behaviour. We chemogenetically activated selective midbrain DA neuronal subpopulations and assessed the effects on feeding microstructure in rats., Results: Treatment with the psychostimulant drug amphetamine or the selective DA reuptake inhibitor GBR 12909 significantly suppressed food intake. Selective chemogenetic activation of DA neurons in the ventral tegmental area (VTA) was found to reduce meal size, but had less impact on total food intake. Targeting distinct VTA neuronal pathways revealed that specific activation of the mesolimbic pathway towards nucleus accumbens (NAc) resulted in smaller and shorter meals. In addition, the meal frequency was increased, rendering total food intake unaffected. The disrupted feeding patterns following activation of VTA DA neurons or VTA to NAc projection neurons were accompanied by locomotor hyperactivity. Activation of VTA neurons projecting towards prefrontal cortex or amygdala, or of DA neurons in the substantia nigra, did not affect feeding behaviour., Conclusions: Chemogenetic activation of VTA DA neurons or VTA to NAc pathway disrupts feeding patterns. Increased activity of mesolimbic DA neurons appears to both promote and reduce food intake, by facilitating both the initiation and cessation of feeding behaviour.

Altered mesolimbic dopamine signaling has been widely implicated in addictive behavior. For the most part, this work has focused on dopamine within the striatum, but there is emerging evidence for a role of the auto-inhibitory, somatodendritic dopamine D2 receptor (D2R) in the ventral tegmental area (VTA) in addiction. Thus, decreased midbrain D2R expression has been implicated in addiction in humans. Moreover, knockout of the gene encoding the D2R receptor (Drd2) in dopamine neurons has been shown to enhance the locomotor response to cocaine in mice. Therefore, we here tested the hypothesis that decreasing D2R expression in the VTA of adult rats, using shRNA knockdown, promotes addiction-like behavior in rats responding for cocaine or palatable food. Rats with decreased VTA D2R expression showed markedly increased motivation for both sucrose and cocaine under a progressive ratio schedule of reinforcement, but the acquisition or maintenance of cocaine self-administration were not affected. They also displayed enhanced cocaine-induced locomotor activity, but no change in basal locomotion. This robust increase in incentive motivation was behaviorally specific, as we did not observe any differences in fixed ratio responding, extinction responding, reinstatement or conditioned suppression of cocaine, and sucrose seeking. We conclude that VTA D2R knockdown results in increased incentive motivation, but does not directly promote other aspects of addiction-like behavior.

Single nucleotide polymorphisms (SNPs) clustered in the first intron of the fat mass and obesity-associated (FTO) gene has been associated with obesity. FTO expression is ubiquitous, with particularly high levels in the hypothalamic area of the brain. To investigate the region-specific role of FTO, AAV technology was applied to knockdown FTO in the ventromedial hypothalamus (VMH). No effect of FTO knockdown was observed on bodyweight or parameters of energy balance. Animals were exposed twice to an overnight fast, followed by a high-fat high-sucrose (HFHS) diet for 1 week. FTO knockdown did not result in a different response to the diets. A region-specific role for FTO in the VMH in the regulation of energy balance could not be found., (© 2014 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.)

We here describe a technique to transiently activate specific neural pathways in vivo. It comprises the combined use of a CRE-recombinase expressing canine adenovirus-2 (CAV-2) and an adeno-associated virus (AAV-hSyn-DIO-hM3D(Gq)-mCherry) that contains the floxed inverted sequence of the designer receptor exclusively activated by designer drugs (DREADD) hM3D(Gq)-mCherry. CAV-2 retrogradely infects projection neurons, which allowed us to specifically express hM3D(Gq)-mCherry in neurons that project from the ventral tegmental area (VTA) to the nucleus accumbens (Acb), the majority of which were dopaminergic. Activation of hM3D(Gq)-mCherry by intraperitoneal (i.p.) injections of clozapine-N-oxide (CNO) leads to increases in neuronal activity, which enabled us to specifically activate VTA to Acb projection neurons. The VTA to Acb pathway is part of the mesolimbic dopamine system and has been implicated in behavioral activation and the exertion of effort. Injections of all doses of CNO led to increases in progressive ratio (PR) performance. The effect of the lowest dose of CNO was suppressed by administration of a DRD1-antagonist, suggesting that CNO-induced increases in PR-performance are at least in part mediated by DRD1-signaling. We hereby validate the combined use of CAV-2 and DREADD-technology to activate specific neural pathways and determine consequent changes in behaviorally relevant paradigms.

The worldwide obesity epidemic poses an enormous and growing threat to public health. However, the neurobehavioral mechanisms of overeating and obesity are incompletely understood. It has been proposed that addiction-like processes may underlie certain forms of obesity, in particular those associated with binge eating disorder. To investigate the role of addiction-like processes in obesity, we adapted a model of cocaine addiction-like behavior in rats responding for highly palatable food. Here, we tested whether rats responding for highly palatable chocolate Ensure would come to show three criteria of addiction-like behavior, i.e., high motivation, continued seeking despite signaled non-availability and persistence of seeking despite aversive consequences. We also investigated whether exposure to a binge model (a diet consisting of alternating periods of limited food access and access to highly palatable food), promotes the appearance of food addiction-like behavior. Our data show substantial individual differences in control over palatable food seeking and taking, but no distinct subgroup of animals showing addiction-like behavior could be identified. Instead, we observed a wide range extending from low to very high control over palatable food intake. Exposure to the binge model did not affect control over palatable food seeking and taking, however. Animals that showed low control over palatable food intake (i.e., scored high on the three criteria for addiction-like behavior) were less sensitive to devaluation of the food reward and more prone to food-induced reinstatement of extinguished responding, indicating that control over palatable food intake is associated with habitual food intake and vulnerability to relapse. In conclusion, we present an animal model to assess control over food seeking and taking. Since diminished control over food intake is a major factor in the development of obesity, understanding its behavioral and neural underpinnings may facilitate improved management of the obesity epidemic.

Food anticipatory activity (FAA) is displayed in rats when access to food is restricted to a specific time frame of their circadian phase, a behavior thought to reflect both hunger and the motivation to eat. Rats also display FAA in a feeding schedule with ad libitum access to normal chow, but limited availability of a palatable meal, which is thought to involve mainly motivational aspects. The orexigenic hormone ghrelin has been implicated in FAA in rodents with restricted access to chow. Because ghrelin plays an important role not only in the control of food intake, but also in reward, we sought to determine the role of ghrelin in anticipation to a palatable meal. Plasma ghrelin levels of non-restricted rats that anticipated chocolate correlated positively with FAA and were increased compared with chow-fed control rats. Furthermore, centrally injected ghrelin increased, whereas an antagonist of the ghrelin receptor decreased, the anticipation to chocolate. Therefore, we hypothesize that central ghrelin signaling is able to mediate the motivational drive to eat.

The dramatically increasing prevalence of obesity, associated with potentially life-threatening health problems, including cardiovascular diseases and type II diabetes, poses an enormous public health problem. It has been proposed that the obesity epidemic can be explained by the concept of 'food addiction'. In this review we focus on possible similarities between binge eating disorder (BED), which is highly prevalent in the obese population, and drug addiction. Indeed, both behavioral and neural similarities between addiction and BED have been demonstrated. Behavioral similarities are reflected in the overlap in DSM-IV criteria for drug addiction with the (suggested) criteria for BED and by food addiction-like behavior in animals after prolonged intermittent access to palatable food. Neural similarities include the overlap in brain regions involved in food and drug craving. Decreased dopamine D2 receptor availability in the striatum has been found in animal models of binge eating, after cocaine self-administration in animals as well as in drug addiction and obesity in humans. To further explore the neurobiological basis of food addiction, it is essential to have an animal model to test the addictive potential of palatable food. A recently developed animal model for drug addiction involves three behavioral characteristics that are based on the DSM-IV criteria: i) extremely high motivation to obtain the drug, ii) difficulty in limiting drug seeking even in periods of explicit non-availability, iii) continuation of drug-seeking despite negative consequences. Indeed, it has been shown that a subgroup of rats, after prolonged cocaine self-administration, scores positive on these three criteria. If food possesses addictive properties, then food-addicted rats should also meet these criteria while searching for and consuming food. In this review we discuss evidence from literature regarding food addiction-like behavior. We also suggest future experiments that could further contribute to our understanding of behavioral and neural commonalities and differences between obesity and drug addiction., (Copyright © 2012 S. Karger GmbH, Freiburg.)

The alarming increase in the incidence of obesity and obesity-associated disorders makes the etiology of obesity a widely studied topic today. As opposed to 'homeostatic feeding', where food intake is restricted to satisfy one's biological needs, the term 'non-homeostatic' feeding refers to eating for pleasure or the trend to over-consume (palatable) food. Overconsumption is considered a crucial factor in the development of obesity. Exaggerated consumption of (palatable) food, coupled to a loss of control over food intake despite awareness of its negative consequences, suggests that overeating may be a form of addiction. At a molecular level, insulin and leptin resistance are hallmarks of obesity. In this review, we specifically address the question how leptin resistance contributes to enhanced craving for (palatable) food. Since dopamine is a key player in the motivation for food, the interconnection between dopamine, leptin and neuropeptides related to feeding will be discussed. Understanding the mechanisms by which these neuropeptidergic systems hijack the homeostatic feeding mechanisms, thus leading to overeating and obesity is the primary aim of this review. The melanocortin system, one of the crucial neuropeptidergic systems modulating feeding behavior will be extensively discussed. The inter-relationship between neuronal populations in the arcuate nucleus and other areas regulating energy homeostasis (lateral hypothalamus, paraventricular nucleus, ventromedial hypothalamus etc.) and reward circuitry (the ventral tegmental area and nucleus accumbens) will be evaluated and scrutinized., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: A significant number of asymptomatic newborns infected with congenital cytomegalovirus (CMV) will present with permanent childhood hearing impairment (PCHI) during early childhood., Objectives: To investigate the role of congenital CMV infection in causing PCHI in the Netherlands, and assess the efficacy of two different hearing screening strategies and the developmental outcome following each strategy., Study Design: We included 192 children with PCHI at the age of 3-5 years, who were offered hearing screening in their first year of life. Dried blood spots from 171 children were available for CMV detection using real-time PCR. The results of eight previously tested samples were also available. Clinical baseline characteristics were collected from medical records and the Child Development Inventory was used to investigate the developmental outcome., Results: The rate of congenital CMV among the 179 children was 8% (14/179) and 23% (9/39) among children with profound PCHI. Two of eight CMV-positive children with PCHI at the age of 3-5 years had passed the newborn hearing screening (NHS) test. Developmental outcome measures showed a significantly greater delay in language comprehension in children with both PCHI and congenital CMV infection (the largest in symptomatic children) than in the children with PCHI without congenital CMV infection., Conclusions: Congenital CMV infection is important in the etiology of PCHI. Universal NHS is not a guarantee of normal hearing and development in childhood for children with congenital CMV infection. This is a problem which might be solved by universal congenital CMV screening.

Background: Because of limited treatment options for congenital cytomegalovirus (CMV) infection, preventive strategies are important. Knowledge and awareness are essential for the success of preventive strategies., Objectives: To investigate the knowledge of congenital CMV among doctors involved in mother and child care in the Netherlands., Study Design: A questionnaire on CMV infection was sent to doctors by snowball sampling. Knowledge concerning epidemiology, transmission, symptoms and signs of CMV infection in adults and children, and treatment options were evaluated., Results: The questionnaire was completed by 246 doctors involved in mother and child care. The respondents estimated a prevalence of congenital CMV varying between 0.1 and 500 per 1000 live-born infants. The mean knowledge scores regarding transmission and postnatal symptoms increased with a more advanced career stage (i.e. older age). Gender and parenthood did not contribute to knowledge, but the field of expertise did. Respondents in the field of pediatrics had the highest mean score on postnatal symptoms and long-term effects. Respondents working in the field of gynecology and obstetrics were unaware of the precise transmission route of CMV. More than one-third of the respondents assumed that treatment was readily available for congenital CMV infection., Conclusions: The knowledge of CMV infection among doctors in the Netherlands contained several gaps. Increasing knowledge and awareness is expected to enhance the prevention of transmission, to improve recognition, and to stimulate diagnostic investigations and follow-up programs.

Background: Combining bronchodilators has been shown to be beneficial in patients with COPD. The additive effects of short-acting bronchodilators added to maintenance tiotropium therapy, however, are unknown., Methods: Following 3 weeks of tiotropium pretreatment, 60 patients with COPD (FEV1 40% of predicted) participated in a randomized, placebo-controlled study to assess add-on bronchodilator effects of ipratropium bromide (40 microg) or fenoterol (200 microg). Short-acting bronchodilators were added as a single dose 2 h and 8 h after tiotropium dosing. Serial lung function tests were performed over 9 h., Results: The peak FEV1 add-on response within 6 h with fenoterol was significantly greater than with placebo (137 mL) or ipratropium (84 mL); the response with ipratropium was slightly but significantly larger than with placebo (52 mL). One hour after the second dose of the test drugs, a similar order of treatment responses was found. The peak FVC add-on response was significant for fenoterol (249 mL) but not for ipratropium (42 mL)., Conclusions: In conclusion, both short-acting bronchodilator classes were effective when added to maintenance treatment with tiotropium. The addition of the beta2-adrenergic fenoterol provided greater additional bronchodilatation than the short-acting anticholinergic ipratropium. This is consistent with the expected effects of combining bronchodilators with different pharmacologic mechanisms. This randomized controlled trial was registered at www.clinicaltrials.gov (NCT00274066).

Proper heart function relies on high efficiency of energy conversion. Mitochondrial oxygen-dependent processes transfer most of the chemical energy from metabolic substrates into ATP. Healthy myocardium uses mainly fatty acids as its major energy source, with little contribution of glucose. However, lactate, ketone bodies, amino acids or even acetate can be oxidized under certain circumstances. A complex interplay exists between various substrates responding to energy needs and substrate availability. The relative substrate concentration is the prime factor defining preference and utilization rate. Allosteric enzyme regulation and protein phosphorylation cascades, partially controlled by hormones such as insulin, modulate the concentration effect; together they provide short-term adjustments of cardiac energy metabolism. The expression of metabolic machinery genes is also dynamically regulated in response to developmental and (patho)physiological conditions, leading to long-term adjustments. Specific nuclear receptor transcription factors and co-activators regulate the expression of these genes. These include peroxisome proliferator-activated receptors and their nuclear receptor co-activator, estrogen-related receptor and hypoxia-inducible transcription factor 1. Increasing glucose and reducing fatty acid oxidation by metabolic regulation is already a target for effective drugs used in ischemic heart disease and heart failure. Interaction with genetic factors that control energy metabolism could provide even more powerful pharmacological tools.

There is scant literature about patients in the final phase of dementia. Uniform terminology and operational definition of the final phase of dementia is lacking. Furthermore, it is difficult to monitor these patients because existing assessment scales face bottom- or ceiling effects in this population. The aim of this study was to assess the prevalence and the characteristics of patients in the final phase of dementia in a group of 210 Dutch nursing home patients with dementia. Stage 7 of the Global Deterioration Scale of Reisberg et al. was used to operationally define the final phase of dementia. All patients were scored on a self-constructed assessment scale. Furthermore, treatment aspects and advance directives were registered.

Prevalence and characteristics of a group dementia patients in the final phase of dementia.There is scant literature about patients in the final phase of dementia. Uniform terminology and operational definition of the final phase of dementia is lacking. Furthermore, it is difficult to monitor these patients because existing assessment scales face bottom- or ceiling effects in this population. The aim of this study was to assess the prevalence and the characteristics of patients in the final phase of dementia in a group of 210 Dutch nursing home patients with dementia. Stage 7 of the Global Deterioration Scale of Reisberg et al. was used to operationally define the final phase of dementia. All patients were scored on a self-constructed assessment scale. Furthermore, treatment aspects and advance directives were registered.Twelve percent (26) of the dementia patients admitted to the psychogeriatric wards met the criteria for final phase of dementia. There was considerable variation in the characteristics of the patients. This study gives a start to further research on aspects of the final phase of dementia.

Objective: Cardiac energetics and performance depend on the expression level of the fast (alpha-) and slow (beta-) myosin heavy chain (MHC) isoform. In ventricular tissue, the beta-MHC isoform predominates, whereas in atrial tissue a variable mixture of alpha- and beta-MHC is found. In several cardiac diseases, the slow isoform is upregulated; however, the functional implications of this transition in human myocardium are largely unknown. The aim of this study was to determine the relation between contractile properties and MHC isoform composition in healthy human myocardium using the diversity in atrial tissue., Methods: Isometric force production and ATP consumption were measured in chemically skinned atrial trabeculae and ventricular muscle strips, and rate of force redevelopment was studied using single cardiomyocytes. MHC isoform composition was determined by one-dimensional SDS-gel electrophoresis., Results: Force development in ventricular tissue was about 5-fold more economical, but nine times slower, than in atrial tissue. Significant linear correlations were found between MHC isoform composition, ATP consumption and rate of force redevelopment., Conclusion: These results clearly indicate that even a minor shift in MHC isoform expression has considerable impact on cardiac performance in human tissue.

We used two experimental models to prove that resveratrol (trans-3,4',5-trihydroxystilbene) reduces cardiac ischemic-reperfusion injury by means of a nitric oxide- and adenosine-dependent mechanism. (1). ACUTE EX VIVO: resveratrol (10 microM, 10 min) infusion in Langendorff-perfused normoxic rat hearts significantly increased adenosine release and coronary flow compared with baseline. After 30-min low-flow ischemia, vasodilation, still present at reperfusion, was completely abolished by resveratrol plus adenosine antagonist 8-(p-sulfophenyl)theophylline (SPT, 50 microM) administration. (2). CHRONIC IN VIVO: rats received tap water containing 25 mg/l resveratrol for 15 days or normal water. Twenty-four hours after, their hearts were Langendorff-perfused and submitted to 60-min low-flow ischemia and reperfusion. The resveratrol-treated hearts showed better functional recovery at reperfusion and significant vasodilation, but no variation in high-energy phosphates (31P Nuclear Magnetic Resonance). N(G)-nitro-L-arginine methyl ester (L-NAME, 30 microM), a nonselective nitric oxide synthase inhibitor, or SPT (50 microM) administered for 10 min prior to the low-flow ischemia cancelled the effects. This suggests that long-term moderate resveratrol consumption could play an important role in late cardioprotective effects.

Objective: Phosphorylation of the myosin light chain 2 (MLC-2) isoform expressed as a percentage of total MLC-2 was decreased in failing (21.1+/-2.0%) compared to donor (31.9+/-4.8%) hearts. To assess the functional implications of this change, we compared the effects of MLC-2 dephosphorylation on force development in failing and non-failing (donor) human hearts., Methods: Cooperative effects in isometric force and rate of force redevelopment (K(tr)) were studied in single Triton-skinned human cardiomyocytes at various [Ca(2+)] before and after protein phosphatase-1 (PP-1) incubation., Results: Maximum force and K(tr) values did not differ between failing and donor hearts, but Ca(2+)-sensitivity of force (pCa(50)) was significantly higher in failing myocardium (Deltap Ca(50)=0.17). K(tr) decreased with decreasing [Ca(2+)], although this decrease was less in failing than in donor hearts. Incubation of the myocytes with PP-1 (0.5 U/ml; 60 min) decreased pCa(50) to a larger extent in failing (0.20 pCa units) than in donor cardiomyocytes (0.10 pCa units). A decrease in absolute K(tr) values was found after PP-1 in failing and donor myocytes, while the shape of the K(tr)-Ca(2+) relationships remained unaltered., Conclusions: Surprisingly, the contractile response to MLC-2 dephosphorylation is enhanced in failing hearts, despite the reduced level of basal MLC-2 phosphorylation. The enhanced response to MLC-2 dephosphorylation in failing myocytes might result from differences in basal phosphorylation of other thin and thick filament proteins between donor and failing hearts. Regulation of Ca(2+)-sensitivity via MLC-2 phosphorylation may be a potential compensatory mechanism to reverse the detrimental effects of increased Ca(2+)-sensitivity and impaired Ca(2+)-handling on diastolic function in human heart failure.

Objective: The alterations in contractile proteins underlying enhanced Ca(2+)-sensitivity of the contractile apparatus in end-stage failing human myocardium are still not resolved. In the present study an attempt was made to reveal to what extent protein alterations contribute to the increased Ca(2+)-responsiveness in human heart failure., Methods: Isometric force and its Ca(2+)-sensitivity were studied in single left ventricular myocytes from non-failing donor (n=6) and end-stage failing (n=10) hearts. To elucidate which protein alterations contribute to the increased Ca(2+)-responsiveness isoform composition and phosphorylation status of contractile proteins were analysed by one- and two-dimensional gel electrophoresis and Western immunoblotting., Results: Maximal tension did not differ between myocytes obtained from donor and failing hearts, while Ca(2+)-sensitivity of the contractile apparatus (pCa(50)) was significantly higher in failing myocardium (deltapCa(50)=0.17). Protein analysis indicated that neither re-expression of atrial light chain 1 and fetal troponin T (TnT) nor degradation of myosin light chains and troponin I (TnI) are responsible for the observed increase in Ca(2+)-responsiveness. An inverse correlation was found between pCa(50) and percentage of phosphorylated myosin light chain 2 (MLC-2), while phosphorylation of MLC-1 and TnT did not differ between donor and failing hearts. Incubation of myocytes with protein kinase A decreased Ca(2+)-sensitivity to a larger extent in failing (deltapCa(50)=0.20) than in donor (deltapCa(50)=0.03) myocytes, abolishing the difference in Ca(2+)-responsiveness. An increased percentage of dephosphorylated TnI was found in failing hearts, which significantly correlated with the enhanced Ca(2+)-responsiveness., Conclusions: The increased Ca(2+)-responsiveness of the contractile apparatus in end-stage failing human hearts cannot be explained by a shift in contractile protein isoforms, but results from the complex interplay between changes in the phosphorylation status of MLC-2 and TnI.

The increased Ca(2+)-responsiveness in end-stage human heart failure cannot be attributed to contractile protein isoform changes, but rather is the complex resultant of changes in degree of phosphorylation of VLC-2 and TnI. Despite the decreased basal level of VLC-2 phosphorylation the response to VLC-2 dephosphorylation is enhanced in failing myocytes, which might result from differences in endogenous phosphorylation of thin and thick filament proteins between donor and failing hearts. Taken together decreased VLC-2 phosphorylation in end-stage human heart failure might represent a compensatory process leading to an improvement of myocardial contractility by opposing the detrimental effects of increased Ca(2+)-responsiveness of force and impaired Ca(2+)-handling on diastolic function.

Our study in rat hearts examined whether activation of adenosine A(1) or A(3) receptors improved functional recovery and reduced apoptosis resulting from low-flow ischemia. Prior to 30 min low-flow ischemia (0.6 ml/min; 6% of baseline flow), Langendorff rat hearts were preconditioned with two 5-min cycles of (a) ischemia (PC; n=7), (b) infusion of 250 nM adenosine A(1) receptor agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA; n=6), or (c) infusion of 50 nM adenosine A(3) receptor agonist N(6)-(3-iodobenzyl)-adenosine-5'-N-methyl-uronamide (IB-MECA; n=8). Recovery of function was improved in PC (71+/-3%), CCPA (68+/-6%) and IB-MECA (68+/-4%) groups compared to control hearts (46+/-5%; P<0.05). Cumulative release of total purines during ischemia-reperfusion was approx. 50% lower in PC, CCPA and IB-MECA groups compared to controls (P<0.05) and was significantly correlated to the percentage functional recovery (R(2)=0.55; P<0.05). The number of cytosolic histone-associated-DNA fragments, a hallmark of apoptosis and measured by Enzyme Linked ImmunoSorbent Assay (ELISA), was small and not different between groups after 30 min reperfusion. However, CCPA (0.6+/-0.1 absorbance units) and MECA (0.7+/-0.1 units; P<0.05 vs. PC) decreased apoptosis after 150 min reperfusion compared to PC (1.4+/-0.3 units) and control (1.2+/-0.1 units) hearts. This study shows that adenosine triggers protection of function in preconditioned rat hearts via both the adenosine A(1) and A(3) receptor. In clinical practice, pharmacological stimulation of adenosine A(3) receptors may be advantageous over adenosine A(1) receptor activation due to a lack of contractile side-effects. In contrast to ischemic preconditioning, pharmacological stimulation of adenosine A(1) or A(3) receptors reduced apoptosis. Furthermore, total purine release may serve as a marker of the degree of functional protection.

In failing human myocardium changes occur, in particular, in isoform composition and phosphorylation level of the troponin T (TnT) and troponin I (TnI) subunits of the actin filament and the myosin light chains (MLC-1 and -2), but it is unclear to what extent they influence cardiac performance. This overview concentrates on the relation between contractile function, contractile protein composition and phosphorylation levels in small biopsies from control (donor) hearts, from biopsies obtained during open heart surgery (NYHA Class I-IV) and from end-stage failing (explanted, NYHA class IV) hearts. Furthermore, attention is paid to the effect of the catalytic subunit of protein kinase A on isometric force development in single Triton-skinned human cardiomyocytes isolated from donor and end-stage failing left ventricular myocardium at different resting sarcomere lengths. A reduction in sarcomere length from 2.2 to 1.8 microm caused reductions in maximum isometric force by approximately 35% both in donor and in failing cardiomyocytes. The midpoints of the calcium sensitivity curves (pCa50) of donor and end-stage failing hearts differed markedly at all sarcomere lengths (mean delta pCa50 = 0.22). Our findings indicate that 1) TnI phosphorylation contributes to the differences in calcium sensitivity between donor and end-stage failing hearts, 2) human ventricular myocardium is heterogeneous with respect of the phosphorylation of TnT, MLC-2 and the isoform distribution of MLC-1 and MLC-2, and 3) the Frank-Starling mechanism is preserved in end-stage failing myocardium.

We tested the hypothesis that down-regulated hearts, as observed during low-flow ischemia, adapt better to low O2 supply than non-down-regulated, or hypoxic, hearts. To address the link between down-regulation and endogenous ischemic protection, we compared myocardial tolerance to ischemia and hypoxia of increasing duration. To that end, we exposed buffer-perfused rat hearts to either low-flow ischemia or hypoxia (same O2 shortage) for 20, 40 or 60 min (n = 8/group), followed by reperfusion or reoxygenation (20 min, full O2 supply). At the end of the O2 shortage, the rate-pressure product was less in ischemic than hypoxic hearts (p < 0.0001). The recovery of the rate-pressure product after reperfusion or reoxygenation was not different for t = 20 min, but was better in ischemic than hypoxic hearts for t = 40 and 60 min (p < 0.02 and p < 0.0002, respectively). The end-diastolic pressure remained unchanged during low-flow ischemia (0.024 +/- 0.013 mmHg x min(-1)), but increased significantly during hypoxia (0.334 +/- 0.079 mmHg x min(-1)). We conclude that, while the duration of hypoxia progressively impaired the rate-pressure product and the end-diastolic pressure, hearts were insensitive of the duration of low-flow ischemia, thereby providing evidence that myocardial down-regulation protects hearts from injury. Excessive ATP catabolism during ischemia in non-down-regulated hearts impaired myocardial recovery regardless of vascular, blood-related and neuro-hormonal factors. These observations support the view that protection is mediated by the maintenance of the ATP pool.

Background: During ischemia, the intracellular calcium and inorganic phosphate (P(i)) concentrations rise and pH falls. We investigated the effects of these changes on force development in donor and failing human hearts to determine if altered contractile protein composition during heart failure changes the myocardial response to Ca(2+), P(i), and pH., Methods and Results: Isometric force was studied in mechanically isolated Triton-skinned single myocytes from left ventricular myocardium. Force declined with added P(i) to 0.33+/-0.02 of the control force (pH 7.1, 0 mmol/L P(i)) at 30 mmol/L P(i) and increased with pH from 0.64+/-0.03 at pH 6.2 to 1.27+/-0.02 at pH 7.4. Force dependency on P(i) and pH did not differ between donor and failing hearts. Incubation of myocytes in a P(i)-containing activating solution caused a potentiation of force, which was larger at submaximal than at maximal [Ca(2+)]. Ca(2+) sensitivity of force was similar in donor hearts and hearts with moderate cardiac disease, but in end-stage failing myocardium it was significantly increased. The degree of myosin light chain 2 phosphorylation was significantly decreased in end-stage failing compared with donor myocardium, resulting in an inverse correlation between Ca(2+) responsiveness of force and myosin light chain 2 phosphorylation., Conclusions: Our results indicate that contractile protein alterations in human end-stage heart failure alter Ca(2+) responsiveness of force but do not affect the force-generating capacity of the cross-bridges or its P(i) and pH dependence. In end-stage failing myocardium, the reduction in force by changes in pH and [P(i)] at submaximal [Ca(2+)] may even be less than in donor hearts because of the increased Ca(2+) responsiveness.

Cardioprotection by K(ATP) channel openers during ischemia is well documented although ill understood. Proarrhythmic effects may be an important drawback. K(ATP) channel modulation influences neurotransmitter release during ischemia in brain synaptosomes. Therefore, we studied the effects of K(ATP) channel modulation on myocardial noradrenaline release and arrhythmias in ischemic rabbit hearts. Isolated rabbit hearts were perfused according to Langendorff and stimulated. Local electrograms were recorded and K+-selective electrodes were inserted in the left ventricular free wall. Cromakalim (3 microM) or glibenclamide (3 microM) was added 20 min prior to induction of global ischemia. After 15, 20, or 30 min of ischemia, hearts were reperfused and noradrenaline content of the first 100 ml of reperfusate was measured. Cromakalim (n = 16) prevented the second rise of extracellular [K(+)] in accordance with its cardioprotective effect. Cromakalim significantly reduced noradrenaline release after 15 min (mean, 169 +/- SEM 97 pmol/gr dry weight vs. control 941 +/- 278; p < 0.05) and 20 min of ischemia (230 +/- 125 pmol/gr dry wt vs. control 1,460 +/- 433; p < 0.05), but after 30 min of ischemia, the difference in noradrenaline release was no longer significant (cromakalim 2,703 +/- 1,195 pmol/gr dry wt vs. control 5,413 +/- 1,310; p = 0.08). Ventricular fibrillation or ventricular tachycardia occurred in 10 of 13 control hearts (77%) (n = 19), in six of 10 glibenclamide-treated hearts (60%) (n = 15), and in six of 14 cromakalim-treated hearts (43%) (p = NS). Cromakalim significantly accelerated onset of ventricular tachycardia or fibrillation (mean +/- SEM onset after 12.5 +/- 1.6 min ischemia vs. control 16.2 +/- 0.7 min; p < 0.05). Noradrenaline release occurred only in cromakalim-treated hearts with early-onset arrhythmias whereas no noradrenaline release was observed in cromakalim-treated hearts without ventricular tachycardia or fibrillation. Our results show that activation of the K(ATP) channel by cromakalim during ischemia reduces myocardial noradrenaline release and postpones the onset of irreversible damage, contributing to the cardioprotective potential of K(ATP) openers during myocardial ischemia.

We tested the hypotheses that (1) progression of coronary artery disease (CAD) increases sympathetic inflow to the heart, thus impairing cardiac blood supply, and (2) reduced sympathetic tone improves cardiac microcirculation and ameliorates severity of anginal symptoms. Electrical irritation of the nerve auricularis--a sensitive ramus of the vagus nerve--provides a central sympatholytic action. Using this technique, we studied the effects of vagal neurostimulation (VNS) on hemodynamics, the content of atrial noradrenergic nerves and the microcirculatory bed of CAD patients. VNS was performed in the preoperative period of CAD patients with severe angina pectoris. The comparison groups consisted of untreated patients with CAD or Wolff-Parkinson-White syndrome. Atrial tissue of patients with this syndrome (n = 6); with effort angina (n = 14); with angina at rest (n = 10); and with severe angina treated with VNS (n = 8) contained the following volume percentages of noradrenergic nerves: 1.7+/-0.1%, 1.3+/-0.3%, 0.5+/-0.1% (p < 0.05 vs. the other groups) and 1.3+/-0.2%, respectively. In these groups, cardiac microcirculatory vessels (diameter, 10-20 microm) had the following densities: 2.7+/-0.2%, 3.4+/-0.2%, 2.0+/-0.4% (p < 0.05 vs. the other groups) and 3.3+/-0.3%, respectively. VNS treatment abolished angina at rest, decreased heart rate and blood pressure. It improved left ventricular ejection fraction from 50+/-1.5% to 58+/-1.0% (p < 0.05), also changing left ventricular diastolic filling. The ratio of time velocity integrals of the early (Ei) to late (Ai) waves increased from 1.07+/-0.12 to 1.65+/-0.17 after VNS (p < 0.05). In electrocardiograms of VNS-treated patients, QRS- and QT-duration were shortened. the PQ-interval did not change, but T-wave configuration improved. In the postoperative period, heart failure occurred in 90% of the control group. vs. 12% in patients treated with VNS (p < 0.05). We conclude that CAD is characterized by overactivity of sympathetic cardiac tone. Vagal stimulation reduced sympathetic inflow to the heart, seemingly via an inhibition of norepinephrine release from sympathetic nerves. VNS' sympatholytic/vagotonic action dilated cardiac microcirculatory vessels and improved left ventricular contractility in patients with severe CAD.

We tested whether ischemic preconditioning of the rat heart is mediated by reduced glycogenolysis during ischemia, an event triggered by adenosine A1 receptor activation. Rat hearts (n=40) were studied with [31P] and [13C] nuclear magnetic resonance (NMR) spectroscopy, using the Langendorff perfusion technique (5.5 mM [1-13C]glucose, 10 U/l insulin). In parallel experiments, hearts (n=43) were freeze-clamped at different time-points throughout the protocol. They were subjected to either ischemic preconditioning (PC), PC in the presence of 50 microM adenosine receptor antagonist, 8-(p-sulfophenyl)-theophylline (SPT), or intermittent infusion of 0.25 microM adenosine A1 receptor agonist, 2-chloro-N6-cyclopentyladenosine (CCPA). After 30 min ischemia and reperfusion, recovery of heart ratexpressure product was improved in hearts treated with preconditioning (33+/-13%) or CCPA (58+/-14%) compared with the SPT and ischemic control (IC) groups, which both failed to recover (P<0.05). CCPA administration induced a 58% increase in pre-ischemic [13C]glycogen (P<0.05 vs. all groups). In the PC and SPT groups, [13C]glycogen decreased by 25 and 47%, respectively (P<0.05) due to the short bouts of ischemia, resulting in lower pre-ischemic glycogen compared to ischemic control and CCPA hearts (P<0.05). The rate of [13C]glycogen utilization during the first 15 min of ischemia (in micromol/min g wwt) was not statistically different between IC (0.42+/-0.03), PC (0.30+/-0.04), and CCPA (0.38+/-0.05) hearts, but was reduced in SPT hearts (0.24+/-0.05; P<0.05). Total glycogen depletion during 30-min ischemia was reduced in PC hearts (0.61 mg/g wwt) compared to IC (1.84 mg/g wwt) and CCPA (1.75 mg/g wwt) hearts; SPT did not block reduced glycogenolysis during ischemia in PC hearts (0.77 mg/g wwt vs. IC). This study adds further strong evidence that in rat hearts, adenosine is involved in ischemic preconditioning. However, protection is unrelated to pre-ischemic glycogen levels and glycogenolysis during ischemia.

Tuberculosis is primarily transmitted from person to person via the respiratory route. We describe five cases of patients who developed tuberculosis at the site of a skin injury: three after being treated repeatedly with local corticosteroids via intramuscular injections, and two who cut themselves accidentally with a knife. All cultures yielded normal-sensitive Mycobacterium tuberculosis, and all patients responded well to anti-tuberculosis treatment. These unusual manifestations of non-respiratory tuberculosis may support the assumption that persistent, painful, reddish and/or fistulous areas of the skin might also indicate an infection caused by M. tuberculosis, via either reactivation of pulmonary tuberculosis or primary infection with M. tuberculosis by cutaneous transmission.

Using (13)C NMR, we tested the hypothesis that protection by preconditioning is associated with reduced glycogenolysis during ischemia. Preconditioned rat hearts showed improved postischemic function and reduced ischemic damage relative to ischemic controls after 30 min stop-flow ischemia and 30 min reperfusion (contractility: 30+/-10 vs. 2+/-2%; creatine kinase release: 41+/-4 vs. 83+/-15 U/g; both P<0.05). Preconditioning decreased preischemic [(13)C]glycogen by 24% (a 10% decrease in total glycogen), and delayed ischemic [(13)C]glycogen consumption by 5-10 min, reducing ischemic glycogenolysis without changing acidosis relative to controls. Upon reperfusion, glycogen synthesis resumed only after preconditioning. Glutamate (13)C-isotopomer analysis showed recovery of Krebs cycle activity with higher anaplerosis than before ischemia (23+/-4 vs. 11+/-3%, P<0.05), but in controls reperfusion failed to restore flux. Compared to control, preconditioning before 20 min ischemia increased contractility (86+/-10 vs. 29+/-14%, P<0.05) and restored preischemic anaplerosis (13+/-3 vs. 39+/-9%, P<0.05). Preconditioning is associated with reduced glycogenolysis early during ischemia. However, protection does not rely on major variations in intracellular pH, as proposed earlier. Our isotopomer data suggest that preconditioning accelerates metabolic and functional recovery during reperfusion by more efficient/active replenishment of the depleted Krebs cycle.