Katharina Bösche

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.

Memory-updating abrogates extinction of learned immunosuppression

When memories are recalled, they enter a transient labile phase in which they can be impaired or enhanced followed by a new stabilization process termed reconsolidation. It is unknown, however, whether reconsolidation is restricted to neurocognitive processes such as fear memories or can be extended to peripheral physiological functions as well. Here, we show in a paradigm of behaviorally conditioned taste aversion in rats memory-updating in learned immunosuppression. The administration of sub-therapeutic doses of the immunosuppressant cyclosporin A together with the conditioned stimulus (CS/saccharin) during retrieval blocked extinction of conditioned taste aversion and learned suppression of T cell cytokine (interleukin-2; interferon-γ) production. This conditioned immunosuppression is of clinical relevance since it significantly prolonged the survival time of heterotopically transplanted heart allografts in rats. Collectively, these findings demonstrate that memories can be updated on both neural and behavioral levels as well as on the level of peripheral physiological systems such as immune functioning.

Extinction of conditioned taste aversion is related to the aversion strength and associated with c-fos expression in the insular cortex

Taste aversion learning is a type of conditioning where animals learn to associate a novel taste (conditioned stimulus; CS) with a stimulus inducing symptoms of poisoning or illness (unconditioned stimulus; US). As a consequence animals later avoid this taste, a reaction known as conditioned taste aversion (CTA). An established CTA extinguishes over time when the CS is repeatedly presented in the absence of the US. However, inter-individual differences in CTA extinction do exist. Using a model of behavioral conditioning with saccharin as CS and the immunosuppressant cyclosporine A as US, the present study aimed at further elucidating the factors underlying individual differences in extinction learning by investigating whether extinction of an established CTA is related to the strength of the initially acquired CS-US association. In addition, we analyzed the expression of the neuronal activation marker c-fos in brain structures relevant for acquisition and retrieval of the CTA, such as the insular cortex and the amygdala. We here show that animals, displaying a strong CS-US association during acquisition, maintained a strong CTA during unreinforced CS re-exposures, in contrast to animals with moderate CS-US association. Moreover, the latter animals showed increased c-fos mRNA expression in the insular cortex. Our data indicate that CTA extinction apparently depends on the strength of the initially learned CS-US association. In addition, these findings provide further evidence that the memory for the initial excitatory conditioning and its subsequent extinction is probably stored in those structures that participate in the processing of the CS and the US.

Exogenous oxytocin reduces signs of sickness behavior and modifies heart rate fluctuations of endotoxemic rats

Besides the well-known roles of oxytocin on birth, maternal bonding, and lactation, recent evidence shows that this hypothalamic hormone possesses cardioprotective, anti-inflammatory and parasympathetic neuromodulation properties. In this study, we explore the heart rate fluctuations (HRF) in an endotoxemic rodent model that was accompanied by the administration of exogenous oxytocin. The assessment of HRF has been widely used as an indirect measure of the cardiac autonomic function. In this context, adult male Dark Agouti rats were equipped with a telemetric transmitter to continuously and remotely measure the electrocardiogram, temperature, and locomotion. In a between-subjects experimental design, rats received the following peripheral treatment: saline solution as a vehicle (V); lipopolysaccharide (LPS); oxytocin (Ox); lipopolysaccharide + oxytocin (LPS+Ox). Linear and non-linear parameters of HRF were estimated starting 3h before to 24h after treatments. Our results showed that exogenous oxytocin does not modify by itself the HRF of oxytocin-treated rats in comparison to vehicle-treated rats. However, in animals undergoing endotoxemia it: a) provokes a less anticorrelated pattern in HRF, b) decreased mean heart rate, c) moderated the magnitude and duration of the LPS-induced hyperthermia, and d) increased locomotion, up to 6h after the LPS injection. The less anticorrelated pattern in the HRF and decreased mean heart rate may reflect a cardiac pacemaker coupling with cholinergic influences mediated by oxytocin during LPS-induced endotoxemia. Finally, the anti-lethargic and long-term temperature moderating effects of the administration of oxytocin during endotoxemia could be a consequence of the systemic anti-inflammatory properties of oxytocin.

Pre-exposure to the unconditioned or conditioned stimulus does not affect learned immunosuppression in rats.

In order to analyze the effects of pre-exposure to either the unconditioned (US) or conditioned stimulus (CS) on learned immunosuppression, we employed an established conditioned taste aversion (CTA) paradigm in rats. In our model, a sweet-tasting drinking solution (saccharin) serves as CS and injection of the immunosuppressive drug cyclosporine A (CsA) is used as US. The conditioned response is reflected by a pronounced CTA and diminished cytokine production by anti-CD3 stimulated splenic T cells. In the present study, experimental animals were exposed either to the US or the CS three times prior to the acquisition phase. On the behavioral level, we found a significantly diminished CTA when animals were pre-exposed to the US or the CS before acquisition. In contrast, US or CS pre-exposure did not affect the behaviorally conditioned suppression of interleukin (IL)-2 production. From the clinical perspective, our data may suggest that conditioning paradigms could be systemically integrated as supportive therapeutic interventions in patients that are already on immunosuppressive therapy or have had previous contact to the gustatory stimulus. Such supportive therapies to pharmacological regimens could not only help to reduce the amount of medication needed and, thus, unwanted toxic side effects, but may also maximize the therapeutic outcome.

Transient inhibition of protein synthesis in the rat insular cortex delays extinction of conditioned taste aversion with cyclosporine A

Conditioned responses gradually weaken and eventually disappear when subjects are repeatedly exposed to the conditioned stimulus (CS) in the absence of the unconditioned stimulus (US), a process called extinction. Studies have demonstrated that extinction of conditioned taste aversion (CTA) can be prevented by interfering with protein synthesis in the insular cortex (IC). However, it remained unknown whether it is possible to pharmacologically stabilize the taste aversive memory trace over longer periods of time. Thus, the present study aimed at investigating the time frame during which extinction of CTA can be efficiently prevented by blocking protein synthesis in the IC. Employing an established conditioning paradigm in rats with saccharin as CS, and the immunosuppressant cyclosporine A (CsA) as US, we show here that daily bilateral intra-insular injections of the protein synthesis inhibitor anisomycin (120μg/μl) immediately after retrieval significantly diminished CTA extinction over a period of five retrieval days and subsequently reached levels of saline-infused controls. These findings demonstrate that it is possible to efficiently delay but not to fully prevent CTA extinction during repeated retrieval trials by blocking protein translation with daily bilateral infusions of anisomycin in the IC. These data confirm and extent earlier reports indicating that the role of protein synthesis in CTA extinction learning is not limited to gastrointestinal malaise-inducing drugs such as lithium chloride (LiCl).

Rats taste-aversive learning with cyclosporine a is not affected by contextual changes.

In conditioned taste aversion (CTA) rats associate a novel taste (conditioned stimulus; CS) with a treatment (unconditioned stimulus; US) that induces symptoms of malaise. During retrieval, animals learn that the CS no longer predicts the US, with the consequence that the behavior elicited by the CS extinguishes. Importantly, CTA data with lithium chloride (LiCl) as US indicate that extinction learning is affected by changing the physical context. However, if this is also the case in different taste-aversion paradigms employing compounds other than LiCL as US is unknown. Against this background the present study investigated in a CTA paradigm with saccharin as CS and the immunosuppressant cyclosporine A (CsA) as US the influence of contextual changes on CTA extinction. Our results show, that extinction of a learned CS-US association with CsA is not prone to contextual changes. Due to the direct effects of CsA on CNS functioning, CTA with this immunosuppressant apparently operates under different mechanisms compared to other drugs, such as LiCl. These data indicate that taste aversive learning and its extinction are not necessarily specific to the context in which it is learned but also depends, at least in part, on the physiological and neuropharmacological effects of the drug employed as US.

Behaviorally conditioned immunosuppression: Effects of pre-exposure to the unconditioned (US) and conditioned (CS) stimulus

We established a model of behaviorally conditioned immunosuppression employing a conditioned taste aversion (CTA) paradigm in rats, pairing a novel taste (saccharin) as a conditioned stimulus (CS) with the immunosuppressive drug cyclosporine A (CsA) as unconditioned stimulus (US). By re-presenting the CS during evocation, rats avoid drinking the saccharin and concomitantly displaying immunosuppression reflected by an reduction in splenic T cell IL-2 production. However, for a possible application of this specific learning protocol in clinical conditions, it is essential to know, whether conditioned immunosuppression could be induced in patients who are already on immunosuppressive therapy or have had previous contact to the gustatory stimulus used as CS. Thus, prior to acquisition rats were exposed three times either to the CS (saccharin) or to the US (CsA). Pre-exposure to both, the US as well as the CS accelerated extinction of the conditioned response on the behavioral level (CTA). Importantly, the conditioned suppression of peripheral immune functioning, reflected as diminished anti-CD3 stimulated IL-2 production, was neither affected by pre-exposure to the US (CsA) nor by the CS (saccharin). These results indicate that learning protocols, such as behaviorally conditioned immunosuppression with CsA, may be employed in clinical situations as supportive therapy together with standard pharmacological regimes, with the goal to reduce the amount of drugs and toxic side effects, maximizing the therapeutic outcome for the patient’s benefit.

Teach the T cells: How learning can shape immunity

mouse skin which was reversed by additional noise stress. We observed a similar pattern of mRNA expression by rtPCR. We tested several Chrna7 antibodies but only one (Abcam, ab110851) gave reliable staining. All these data infer that in mast cells and in cutaneous neuro-immune network, NNCS mediators play a key role in the regulation of stress-sensitive inflammatory responses. Further studies are needed to elucidate the underlying mechanisms and the potential of pharmacological intervention.

Pre-exposure to the unconditioned or the conditioned stimulus differentially affect learned immunosuppression in rats

addition, immunohistochemical staining for CD68 was performed to visualize activated microglial cells in serial sections. Results: The DVR of [11C]PK11195 was significantly increased in the NAWM (p b 0.001) and thalamic ROIs (p = 0.027) of SPMS patients compared to the control group. Increased perilesional TSPO-uptake was present in 57% of the chronic T1-lesions in MRI, giving thus in vivo proof of a significant proportion of chronic active lesions among SPMS patients. Autoradiography performed with autopsy samples of MS brain revealed specific TSPO-ligand binding at the plaque edge. Finally, immunohistochemical staining confirmed co-localization of TSPO-ligand binding with activated, CD68-positive microglial cells/ macrophages. Conclusions: PET imaging enables the detection of microglial activation in brains of SPMS patients in vivo. It thus helps to visualize the diffuse pathology associated with activated microglial cells, and the presence of chronic active lesions.