Investigations on the Ability of the Insular Cortex to Process Peripheral Immunosuppression.

The brain and immune system communicate through complex bidirectional pathways, but the specificity by which the brain perceives or even remembers alterations in immune homeostasis is still poorly understood. Recent data revealed that immune-related information under peripheral inflammatory conditions, termed as "immunengram", were represented in specific neuronal ensembles in the insular cortex (IC). Chemogenetic reactivation of these neuronal ensembles was sufficient to retrieve the inflammatory stages, indicating that the brain can store and retrieve specific immune responses. Against this background, the current approach was designed to investigate the ability of the IC to process states of immunosuppression pharmacologically induced by the mechanistic target of rapamycin (mTOR) inhibitor rapamycin. We here show that the IC perceives the initial state of immunosuppression, reflected by increased deep-brain electroencephalography (EEG) activity during acute immunosuppressive drug treatment. Following an experienced period of immunosuppression, though, diminished splenic cytokine production as formerly induced by rapamycin could not be reinstated by nonspecific chemogenetic activation or inhibition of the IC. These findings suggest that the information of a past, or experienced status of pharmacologically induced immunosuppression is not represented in the IC. Together, the present work extends the view of immune-to-brain communication during the states of peripheral immunosuppression and foster the prominent role of the IC for interoception.

Irradiation in a flash: Unique sparing of memory in mice after whole brain irradiation with dose rates above 100Gy/s.

This study shows for the first time that normal brain tissue toxicities after WBI can be reduced with increased dose rate. Spatial memory is preserved after WBI with mean dose rates above 100Gy/s, whereas 10Gy WBI at a conventional radiotherapy dose rate (0.1Gy/s) totally impairs spatial memory.

Acute systemic rapamycin induces neurobehavioral alterations in rats.

Rapamycin is a drug with antiproliferative and immunosuppressive properties, widely used for prevention of acute graft rejection and cancer therapy. It specifically inhibits the activity of the mammalian target of rapamycin (mTOR), a protein kinase known to play an important role in cell growth, proliferation and antibody production. Clinical observations show that patients undergoing therapy with immunosuppressive drugs frequently suffer from affective disorders such as anxiety or depression. However, whether these symptoms are attributed to the action of the distinct compounds remains rather elusive. The present study investigated in rats neurobehavioral consequences of acute rapamycin treatment. Systemic administration of a single low dose rapamycin (3mg/kg) led to enhanced neuronal activity in the amygdala analyzed by intracerebral electroencephalography and FOS protein expression 90min after drug injection. Moreover, behavioral investigations revealed a rapamycin-induced increase in anxiety-related behaviors in the elevated plus-maze and in the open-field. The behavioral alterations correlated to enhanced amygdaloid expression of KLK8 and FKBP51, proteins that have been implicated in the development of anxiety and depression. Together, these results demonstrate that acute blockade of mTOR signaling by acute rapamycin administration not only causes changes in neuronal activity, but also leads to elevated protein expression in protein kinase pathways others than mTOR, contributing to the development of anxiety-like behavior. Given the pivotal role of the amygdala in mood regulation, associative learning, and modulation of cognitive functions, our findings raise the question whether therapy with rapamycin may induce alterations in patients neuropsychological functioning.

Neurobehavioural activation during peripheral immunosuppression.

Like other physiological responses, immune functions are the subject of behavioural conditioning. Conditioned immunosuppression can be induced by contingently pairing a novel taste with an injection of the immunosuppressant cyclosporine A (CsA) in an associative learning paradigm. This learned immunosuppression is centrally mediated by the insular cortex and the amygdala. However, the afferent mechanisms by which the brain detects CsA are not understood. In this study we analysed whether CsA is sensed via the chemosensitive vagus nerve or whether CsA directly acts on the brain. Our experiments revealed that a single peripheral administration of CsA increases neuronal activity in the insular cortex and the amygdala as evident from increased electric activity, c-Fos expression and amygdaloid noradrenaline release. However, this increased neuronal activity was not affected by prior vagal deafferentation but rather seems to partially be induced by direct action of CsA on cortico-amygdaloid structures and the chemosensitive brainstem regions area postrema and nucleus of the solitary tract. Together, these data indicate that CsA as an unconditioned stimulus may directly act on the brain by a still unknown transduction mechanism.

Amygdaloid signature of peripheral immune activation by bacterial lipopolysaccharide or staphylococcal enterotoxin B.

Activated immune cells produce soluble mediators that not only coordinate local and systemic immune responses but also act on the brain to initiate behavioral, neuroendocrine and metabolic adaptations. Earlier studies have shown that the amygdala, a group of nuclei located in the medial temporal lobe, is engaged in the central processing of afferent signals from the peripheral immune system. Here, we compared amygdaloid responses to lipopolysaccharide (LPS) and staphylococcal enterotoxin B (SEB), two prototypic bacterial products that elicit distinct immune responses. Intraperitoneal administration of LPS (0.1 mg/kg) or SEB (1 mg/kg) in adult rats induced substantial increases in amygdaloid neuronal activity as measured by intracerebral electroencephalography and c-fos gene expression. Amygdaloid neuronal activation was accompanied by an increase in anxiety-related behavior in the elevated plus-maze test. However, only treatment with LPS, but not SEB, enhanced amygdaloid IL-1ß and TNF-α mRNA expression. This supports the view of the immune system as a sensory organ that recognizes invading pathogens and rapidly relays this information to the brain, independent of the nature of the immune response induced. The observation that neuronal and behavioral responses to peripheral immune challenges are not necessarily accompanied by increased brain cytokine expression suggests that cytokines are not the only factors driving sickness-related responses in the CNS.

Acute amygdaloid response to systemic inflammation.

The amygdala, a group of nuclei located in the medial temporal lobe, is a key limbic structure involved in mood regulation, associative learning, and modulation of cognitive functions. Functional neuroanatomical studies suggest that this brain region plays also an important role in the central integration of afferent signals from the peripheral immune system. In the present study, intracerebral electroencephalography and microdialysis were employed to investigate the electrophysiological and neurochemical consequences of systemic immune activation in the amygdala of freely moving rats. Intraperitoneal administration of bacterial lipopolysaccharide (100 µg/kg) induced with a latency of about 2 h a significant increase in amygdaloid neuronal activity and a substantial rise in extracellular noradrenaline levels. Activated neurons in the amygdaloid complex, identified by c-Fos immunohistochemistry, were mainly located in the central nucleus and, to a lesser extent, in the basolateral nucleus of the amygdala. Gene expression analysis in micropunches of the amygdala revealed that endotoxin administration induced a strong time-dependent increase in IL-1ß, IL-6, and TNF-α mRNA levels indicating that these cytokines are de novo synthesized in the amygdala in response to peripheral immune activation. The changes in amygdaloid activity were timely related to an increase in anxiety-like behavior and decreased locomotor activity and exploration in the open-field. Taken together, these data give novel insights into different features of the acute amygdaloid response during experimental inflammation and provides further evidence that the amygdala integrates immune-derived information to coordinate behavioral and autonomic responses.

Electrical activity in rat cortico-limbic structures after single or repeated administration of lipopolysaccharide or staphylococcal enterotoxin B.

Immune-to-brain communication is essential for an individual to aptly respond to challenging internal and external environments. However, the specificity by which the central nervous system detects or 'senses' peripheral immune challenges is still poorly understood. In contrast to post-mortem c-Fos mapping, we recorded neural activity in vivo in two specific cortico-limbic regions relevant for processing visceral inputs and associating it with other sensory signalling, the amygdala (Am) and the insular cortex (IC). Adult rats were implanted with deep-brain monopolar electrodes and electrical activity was monitored unilaterally before and after administration of two different immunogens, the T-cell-independent antigen lipopolysaccharide (LPS) or the T-cell-dependent antigen staphylococcal enterotoxin B (SEB). In addition, the neural activity of the same individuals was analysed after single as well as repeated antigen administration, the latter inducing attenuation of the immune response. Body temperature and circulating cytokine levels confirmed the biological activity of the antigens and the success of immunization and desensitization protocols. More importantly, the present data demonstrate that neural activity of the Am and IC is not only specific for the type of immune challenge (LPS versus SEB) but seems to be also sensitive to the different immune state (naive versus desensitization). This indicates that the forebrain expresses specific patterns of electrical activity related to the type of peripheral immune activation as well as to the intensity of the stimulation, substantiating associative learning paradigms employing antigens as unconditioned stimuli. Overall, our data support the view of an intensive immune-to-brain communication, which may have evolved to achieve the complex energetic balance necessary for mounting effective immunity and improved individual adaptability by cognitive functions.

Stimulation of ß2-adrenergic receptors inhibits calcineurin activity in CD4(+) T cells via PKA-AKAP interaction.

The sympathetic nervous system (SNS) is able to modulate immune functions via adrenoceptor-dependent mechanisms. Activation of ß2-adrenergic receptors (AR) on CD4(+) T lymphocytes has been shown to inhibit Th1-cytokine production and cell proliferation. Here, we investigated the role of the calcium/calmodulin-dependent protein phosphatase calcineurin (CaN), a key element of the T cell receptor (TCR)-signaling pathway, in ß2-AR-mediated suppression of T cell function. Purified rat splenic CD4(+) T cells were stimulated with anti-CD3/anti-CD28 in presence or absence of the ß2-AR agonist terbutaline (TERB). Treatment with TERB induced a dose-dependent inhibition of cellular CaN activity, along with a reduction in IL-2 and IFN-γ production, and T cell proliferation. Co-administration of the ß-AR antagonist nadolol abolished these effects. Blockade of the cAMP-dependent protein kinase A (PKA) with the inhibitor H-89 completely prevented TERB-induced CaN inhibition. However, a receptor-independent rise in the second messenger cAMP was not sufficient to suppress CaN activity. Disruption of the interaction between PKA and A-kinase anchoring protein (AKAP) by the inhibitor peptide St-Ht31 fully blocked TERB-induced CaN inhibition, demonstrating that PKA-AKAP interaction is essential for the ß2-AR-mediated CaN inhibition. Taken together, this study provides evidence for a link between the ß2-AR and TCR signaling pathways since expression of IL-2 and IFN-γ in activated T cells largely depends on dephosphorylation of the transcription factor NFAT by CaN, and identifies a novel intracellular mechanism that can lead to downregulation of T cell function after SNS activation.

The metabolic footprint of aging in mice.

Aging is characterized by a general decline in cellular function, which ultimately will affect whole body homeostasis. Although DNA damage and oxidative stress all contribute to aging, metabolic dysfunction is a common hallmark of aging at least in invertebrates. Since a comprehensive overview of metabolic changes in otherwise healthy aging mammals is lacking, we here compared metabolic parameters of young and 2 year old mice. We systemically integrated in vivo phenotyping with gene expression, biochemical analysis, and metabolomics, thereby identifying a distinguishing metabolic footprint of aging. Among the affected pathways in both liver and muscle we found glucose and fatty acid metabolism, and redox homeostasis. These alterations translated in decreased long chain acylcarnitines and increased free fatty acid levels and a marked reduction in various amino acids in the plasma of aged mice. As such, these metabolites serve as biomarkers for aging and healthspan.

Chemical destruction of brain noradrenergic neurons affects splenic cytokine production.

The neurotransmitter noradrenaline (NA) plays a pivotal role in immune regulation. Here we used the selective neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) to investigate the impact of central NA depletion on cytokine production by splenic monocytes/macrophages and T cells. Intraperitoneal administration of DSP-4 in adult rats induced a substantial reduction of noradrenergic neurons in the locus coeruleus and the A5 cell group. The degeneration of brainstem noradrenergic neurons was accompanied by a significant decrease in the production of interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-alpha by lipopolysaccharide-stimulated splenocytes. In addition, upon T cell receptor stimulation with anti-CD3, isolated splenocytes of DSP-4 treated animals produced significantly less interferon (IFN)-gamma but not IL-2 and IL-4. The proportion of monocytes/macrophages and T cells in the spleen remained unaffected by the neurotoxin treatment, however, the percentage of natural killer cells decreased significantly. The findings suggest that a certain level of central noradrenergic tone is required for normal functioning of peripheral immune cells.

Time-dependent alterations of peripheral immune parameters after nigrostriatal dopamine depletion in a rat model of Parkinson's disease.

Dysfunction of the central dopaminergic system is associated with neurodegenerative disorders and mental illnesses such as Parkinson's disease and schizophrenia. Patients suffering from these diseases were reported to exhibit altered immune functions compared to healthy subjects and imbalance of the central dopaminergic system has been suggested as one causative factor for the immune disturbances. However, it is unclear whether the observed immune changes are primary or secondary to the disease. Here we demonstrate that central dopamine (DA) depletion in a rat model of Parkinson's disease induced transient changes in blood leukocyte distribution and cytokine production that were apparent until four weeks after bilateral intrastriatal administration of the neurotoxin 6-hydroxydopamine (6-OHDA). Eight weeks after treatment, no differences in blood immune parameters were anymore evident between neurotoxin-treated and control animals. Nevertheless, animals with a widespread damage of dopaminergic neurons in the nigrostriatal system showed an exacerbated pro-inflammatory response following in vivo challenge with bacterial lipopolysaccharide. Our data indicate that peripheral immune perturbations in the early phase after intrastriatal 6-OHDA administration might have been related to the neurodegenerative process itself whereas the increased sensitivity to the inflammatory stimulus seems to have resulted from an impaired dopaminergic control of prolactin (PRL) and corticosterone (CORT) secretion. The findings demonstrate that the brain dopaminergic system is involved in peripheral immune regulation and suggest that central dopaminergic hypoactivity bears the risk of excessive inflammation, e.g., during infection or tissue injury.

Calcineurin inhibition in splenocytes induced by pavlovian conditioning.

Pavlovian conditioning is one of the major neurobiological mechanisms of placebo effects, potentially influencing the course of specific diseases and the response to a pharmacological therapy, such as immunosuppression. In our study with behaviorally conditioned rats, a relevant taste (0.2% saccharin) preceded the application of the immunosuppressive drug cyclosporin A (CsA), a specific calcineurin (CaN) inhibitor. Our results demonstrate that through pavlovian conditioning the particular pharmacological properties of CsA can be transferred to a neutral taste, i.e., CaN activity was inhibited in splenocytes from conditioned rats after reexposure to the gustatory stimulus. Concomitant immune consequences were observed on ex vivo mitogenic challenge (anti-CD3). Particularly, Th1-cytokine, but not Th2-cytokine, production and cell proliferation were impeded. Appropriate pharmacological and behavioral controls certify that all these changes in T-lymphocyte reactivity are attributable to mere taste reexposure. Furthermore, the underlying sympathetic-lymphocyte interaction was revealed modeling the conditioned response in vitro. CaN activity in CD4(+) T lymphocytes is reduced by beta-adrenergic stimulation (terbutaline), with these effects antagonized by the beta-adrenoreceptor antagonist nadolol. In summary, CaN was identified as the intracellular target for inducing conditioned immunosuppression by CsA, contributing to our understanding of the intracellular mechanisms behind "learned placebo effects."

Behavioural conditioning of immune functions: how the central nervous system controls peripheral immune responses by evoking associative learning processes.

During the last 30 years of psychoneuroimmunology research the intense bi-directional communication between the central nervous system (CNS) and the immune system has been demonstrated in studies on the interaction between the nervous-endocrine-immune systems. One of the most intriguing examples of such interaction is the capability of the CNS to associate an immune status with specific environmental stimuli. In this review, we systematically summarize experimental evidence demonstrating the behavioural conditioning of peripheral immune functions. In particular, we focus on the mechanisms underlying the behavioural conditioning process and provide a theoretical framework that indicates the potential feasibility of behaviourally conditioned immune changes in clinical situations.