Taste‐Associative Learning in Rats: Conditioned Immunosuppression with Cyclosporine A to Study the Neuro‐Immune Network

Abstract

The pharmacological effects of an immunosuppressive drug, such as cyclosporine A (CsA), can be learned and retrieved by humans and animals when applying associative learning paradigms. This principle is based on Pavlovian conditioning, in which repeated presentation of an "unconditioned stimulus" (US; here, the drug CsA) is paired with exposure to a "conditioned stimulus" (CS; here, the novel taste of saccharin). Re-exposure to the CS at a later time leads to an avoidance behavior. Concomitantly, using this paradigm, animals exposed to the CS (saccharin) display immunosuppression, reflected by reduced splenic T-cell proliferation and diminished interleukin-2 and interferonγ expression and release in ex vivo cultured splenocytes, mimicking the pharmacological effects of the US (CsA). Notably, this paradigm of taste-immune associative learning demonstrates the impressive abilities of the brain to detect and store information about an organism's immunological status and to retrieve this information, thereby modulating immunological functions via endogenous pathways. Moreover, conditioned pharmacological effects, obtained by means of associative learning, have been successfully implemented as controlled drugdose reduction strategies as a supportive treatment option to optimize pharmacological treatment effects for patients' benefit. However, our knowledge about the underlying neurobiological and immunological mechanisms mediating such learned immunomodulatory effects is still limited. A reliable animal model of taste-immune associative learning can provide novel insights into peripheral and central nervous processes. In this article, we describe protocols that focus on the basic taste-immune associative learning paradigm with CsA and saccharin in rats, where conditioned peripheral immunosuppression is determined in ex vivo cultured splenocytes. The behavioral protocol is reliable and adaptable and may pave the road for future studies using taste-immune associative learning paradigms to gain deeper insight into brain-to-immune-system communication.

The Effects of Fingolimod (FTY720) on Leukocyte Subset Circulation cannot be Behaviourally Conditioned in Rats

“…Previous work has demonstrated that paradigms of taste-immune associative learning in rodents using immunomodulatory substances such as CY, CsA or rapamycin as US can induce learned immune responses mimicking those of the pharmacological drug (Ader and Cohen 1975 ; von Horsten et al 1998a ; Niemi et al 2006 ; Lückemann et al 2019 ; Hadamitzky et al 2020 ; Leisengang et al 2022 ). These behaviourally conditioned immune responses are of clinical relevance since they ameliorated symptomatology of autoimmune diseases, prolonged survival of transplanted organs, and prevented tumor growth in pre-clinical studies (Klosterhalfen and Klosterhalfen 1983 ; Grochowicz et al 1991 ; Hadamitzky et al 2016 ; Lückemann et al 2020 ; Hetze et al 2022 ).…”

“…Conditioning was performed based on an established conditioning paradigm (Lückemann et al 2019 ; Leisengang et al 2022 ) with slight modifications. According to specific pharmacokinetics of FTY720 (i.e., long half-life (Kovarik et al 2004 ), the retention interval between acquisition and retrieval was prolonged to 21 days (Fig.…”

“…More importantly, CS re-exposure evokes learned alterations of immune functions, similar to those formerly induced by the immunomodulatory drug administered as US (Hadamitzky et al 2016 ; Hörbelt et al 2019 ; Lückemann et al 2019 ). Employing a taste-immune associative learning paradigm, the immunosuppressive properties of substances such as cyclophosphamide (CY), the calcineurin inhibitor cyclosporine A (CsA) as well as the anti-proliferative capacities of the mechanistic target of rapamycin (mTOR) antagonist rapamycin have already been successfully conditioned in rats (Ader and Cohen 1975 ; von Horsten et al 1998a ; Niemi et al 2006 ; Lückemann et al 2019 ; Hadamitzky et al 2020 ; Leisengang et al 2022 ). These learned immune responses are of clinical relevance since they attenuated the progression of autoimmune diseases, prolonged survival of transplanted solid organs and reduced tumor growth in pre-clinical studies (Klosterhalfen and Klosterhalfen 1983 ; Grochowicz et al 1991 ; Lückemann et al 2020 ; Hetze et al 2022 ).…”