Behavioral Conditioning of Immune Responses with Cyclosporine A in a Murine Model of Experimental Autoimmune Uveitis.

OBJECTIVE: We examined the role of behavioral conditioning of immune responses with cyclosporine A (CsA) on the development of Th1/Th17-driven experimental autoimmune uveoretinitis (EAU). METHODS: Mice received a 0.2% w/v saccharin solution as conditioned stimulus combined with CsA (20 mg/kg) in 6 association trials at 72-h intervals. For evocation periods, conditioned mice were reexposed to saccharin, whereas the conditioned but not reexposed group received water only. Animals were immunized with human interphotoreceptor-retinoid-binding protein peptide 161-180 (hIRBPp161-180) peptide in complete Freund adjuvant (CFA) and a concomitant injection of pertussis toxin. RESULTS: In naïve mice subjected to the behavioral conditioning regimen, mitogen-induced interleukin (IL)-2 production was decreased in conditioned mice compared to conditioned but not reexposed animals. Incidence and severity of EAU were not significantly lower in behaviorally conditioned and immunized mice. ELISA analysis of splenocytes revealed a reduced interferon (IFN)-γ/IL-17 ratio in CsA-treated, conditioned but not reexposed, and conditioned animals. The adoptive transfer of antigen-specific splenocytes from animals behaviorally conditioned with CsA to naïve mice decreased the severity of EAU in recipient mice compared to the control group. In vitro activation of splenocytes isolated from immunized mice with agonists targeting TLR2 and NOD2 together with ß2-adrenergic activation (induced by epinephrine, norepinephrine, or salbutamol) resulted in decreased IFN-γ but increased IL-17 immune responses. The ß2-adrenergic antagonist propranolol could restore IFN-γ production, whereas only the norepinephrine-induced increase in IL-17 production was abrogated. CONCLUSIONS: We conclude that CsA conditioning in the EAU model mitigates Th1 but enhances Th17 immune responses, and does not ameliorate disease. The results imply that in EAU the mechanism of immune conditioning interacts with CFA components during active immunization, most likely via the TLR2/NOD2 pathway, and induces differentiation of Th17 cells that drive autoimmune diseases.

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).

Active vision in marmosets: a model system for visual neuroscience.

The common marmoset (Callithrix jacchus), a small-bodied New World primate, offers several advantages to complement vision research in larger primates. Studies in the anesthetized marmoset have detailed the anatomy and physiology of their visual system (Rosa et al., 2009) while studies of auditory and vocal processing have established their utility for awake and behaving neurophysiological investigations (Lu et al., 2001a,b; Eliades and Wang, 2008a,b; Osmanski and Wang, 2011; Remington et al., 2012). However, a critical unknown is whether marmosets can perform visual tasks under head restraint. This has been essential for studies in macaques, enabling both accurate eye tracking and head stabilization for neurophysiology. In one set of experiments we compared the free viewing behavior of head-fixed marmosets to that of macaques, and found that their saccadic behavior is comparable across a number of saccade metrics and that saccades target similar regions of interest including faces. In a second set of experiments we applied behavioral conditioning techniques to determine whether the marmoset could control fixation for liquid reward. Two marmosets could fixate a central point and ignore peripheral flashing stimuli, as needed for receptive field mapping. Both marmosets also performed an orientation discrimination task, exhibiting a saturating psychometric function with reliable performance and shorter reaction times for easier discriminations. These data suggest that the marmoset is a viable model for studies of active vision and its underlying neural mechanisms.

The click is not the trick: the efficacy of clickers and other reinforcement methods in training naïve dogs to perform new tasks.

BACKGROUND: A handheld metal noisemaker known as a "clicker" is widely used to train new behaviors in dogs; however, evidence for their superior efficacy compared to providing solely primary reinforcement or other secondary reinforcers in the acquisition of novel behavior in dogs is largely anecdotal. METHODS: Three experiments were conducted to determine under what circumstances a clicker secondary reinforcer may result in acquisition of a novel behavior more rapidly or to a higher level compared to other readily available reinforcement methods. In Experiment 1, three groups of 30 dogs each were shaped to emit a novel sit and stay behavior of increasing duration with either the delivery of food alone, a verbal stimulus paired with food, or a clicker with food. The group that received only a primary reinforcer reached a significantly higher criterion of training success than the group trained with a verbal secondary reinforcer. Performance of the group experiencing a clicker as a secondary reinforcer was intermediate between the other two groups, but not significantly different from either. In Experiment 2, three groups of 25 dogs each were shaped to emit a nose targeting behavior and then perform that behavior at increasing distances from the experimenter using the same three methods of positive reinforcement as in Experiment 1. No statistically significant differences between the groups were found. In Experiment 3, three groups of 30 dogs each were shaped to emit a nose-targeting behavior upon an array of wooden blocks with task difficulty increasing throughout testing using the same three methods of positive reinforcement as previously tested. No statistically significant differences between the groups were found. RESULTS: Overall, the findings suggest that both primary reinforcement alone as well as a verbal or clicker secondary reinforcer can be used successfully in training a dog to perform a novel behavior, but that no positive reinforcement method demonstrated significantly greater efficacy than any other.

Behaviorally conditioned immunosuppression with cyclosporine A forms long lasting memory trace.

Behaviorally conditioned taste avoidance (CTA) paradigms using the novel taste saccharin as a conditioned stimulus (CS) and the immunosuppressive drug cyclosporine A (CsA) as unconditioned stimulus (US) demonstrate learned suppression of immune functions. However, conditioned immune responses might decrease over time depending on the period between acquisition and retrieval (retention interval). Thus, the present study investigated whether and to what extent prolonged 14- and 30-days retention intervals affect conditioned behavioral (CTA) and immune responses in rats. Our findings demonstrate that conditioned animals displayed a marked CTA after 14 and 30 days upon CS re-exposure compared to control animals. More importantly, the production of the pro-inflammatory cytokine interferon (IFN)-γ was significantly suppressed in ex vivo anti-CD3 stimulated splenocytes of conditioned animals compared to controls at both time points. These findings document that CTA paradigms using the immunosuppressive drug CsA as US form long lasting memory traces of the learned behavioral and immune responses.

Are Adverse Events Induced by the Acute Administration of Calcineurin Inhibitor Cyclosporine A Behaviorally Conditioned in Healthy Male Volunteers?

PURPOSE: The learned immunosuppressive placebo response has been demonstrated in experimental animals, healthy humans, and patients, and is suggested as a therapy for improving immunopharmacologic treatment. It remains unclear, however, whether potential adverse events induced by the drug are also behaviorally conditioned. Employing an established taste-immune learning paradigm in healthy humans using the calcineurin inhibitor and immunosuppressive drug cyclosporine A (CsA) as an unconditioned stimulus, we investigated whether and to what extent perceived adverse events induced by acute CsA administration are behaviorally conditioned. METHODS: A total of 68 healthy male subjects were exposed to the established taste-immune learning paradigm, receiving either placebo or CsA (10 mg/kg) as an unconditioned stimulus, and a novel-tasting drink as a conditioned stimulus. FINDINGS: Subjects repeatedly receiving CsA during acquisition reported significantly more adverse events than did placebo-receiving subjects. However, during reexposure to the conditioned stimulus, the reported adverse events did not differ from those in the placebo control condition. IMPLICATIONS: These data indicate that acute adverse events are not behaviorally conditioned during the learned immunosuppressive response. Our results further strengthen the great potential clinical relevance of employing the learned immunosuppressive placebo response as a therapy to support immunopharmacologic regimens, ultimately aiming to reduce the medical dosages required, thereby minimizing adverse drug events while maximizing the therapeutic benefit in patients. German Clinical Trial Register (www.drks.de) identifier: DRKS00007693.

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.

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.