Characterisation of the neural basis underlying appetitive extinction & renewal in Cacna1c rats

Abstract

Recent studies have revealed impairments in Cacna1c ± heterozygous animals (a gene that encodes the Cav 1.2 L-type voltage-gated calcium channels and is implicated in risk for multiple neuropsychiatric disorders) in aversive forms of learning, such as latent inhibition, reversal learning or context discrimination. However, the role of Cav 1.2 L-type voltage-gated calcium channels in extinction of appetitive associations remains under-investigated. Here, we used an appetitive Pavlovian conditioning task and evaluated extinction learning (EL) with a change of context from that of training and test (ABA) and without such a change (AAA) in Cacna1c ± male rats versus their wild-type (WT) littermates. In addition, we used fluorescence in situ hybridization of somatic immediate early genes (IEGs) Arc and Homer1a expression to scrutinize associated changes in the medial prefrontal cortex and the amygdala. Cacna1c ± animals successfully adapt their responses by engaging in appetitive EL and renewal. However, the regional IEG expression profile changed. For the EL occurring in the same context, Cacna1c ± animals presented higher IEG expression in the infralimbic cortex and the central amygdala than controls. The prelimbic region presented a larger neural ensemble in Cacna1c ± than WT animals, co-labelled for the time window of EL in the original context and prolonged exposure to the unrewarded context. With a context change, the Cacna1c ± infralimbic region displayed higher IEG expression during renewal than controls. Taken together, our findings provide novel evidence of distinct brain activation patterns occurring in Cacna1c ± rats after appetitive extinction and renewal despite preserved behavioral responses.

This article is part of the Special Issue on "L-type calcium channel mechanisms in neuropsychiatric disorders".

Introduction

Synaptic plasticity is a fundamental property of neurons including activity-dependent changes in the efficacy and strength of synaptic transmission at preexisting synapses. Such changes are the major cellular mechanisms underlying learning and memory. Although several types of synaptic plasticity have been delineated across neuronal types and brain regions, all of them share a critical role for Ca²⁺-mediated processes (see Mateos-Aparicio and Rodríguez-Moreno, 2020). In addition, some symptoms of psychosis have been suggested to stem from alterations in associative learning related to changes in synaptic plasticity (Kapur, 2003; Hall et al., 2009). Genome-wide association studies (GWAS) have strongly related genetic variation in CACNA1C, a gene that encodes the Cav 1.2 L-type voltage-gated calcium channels, with an increased risk of psychiatric disorders (Ferreira et al., 2008; Hall et al., 2015). These studies identify single nucleotide polymorphisms (SNPs), associated with bipolar disorder, schizophrenia, major depressive disorder, and autism (Moon et al., 2018; Green et al., 2013). The effect of CACNA1C risk-associated SNPs remains unclear. They produce altered CACNA1C dosage and in some cases decrease CACNA1C expression (e.g., there is good evidence that the risk SNPs rs1006737 is associated with decreased expression in the hippocampus), but the way they impact the gene is not consistent across studies (Bigos et al., 2010; Roussos et al., 2014; Yoshimizu et al., 2015; Tigaret et al., 2021).

CACNA1C expresses the transcript Cav1.2 that plays a key role in learning and memory by modulating learning-related neural pathways. Calcium influx in post-synaptic neurons signals cascades to regulate the activity and transcription factors such as the cAMP response element-binding protein (CREB), nuclear factor of activated T cells (NFTA) pathway, and Hebbian synapse plasticity (Deisseroth et al., 2003; Moosmang et al., 2005). Critically, calcium influx via L-type voltage-gated calcium channels triggers the transcription of calcium-regulated genes including brain-derived neurotrophic factor (BDNF), which has a significant role in learning processes (West et al., 2001). Moreover, recent studies have revealed cognitive deficits in a hemizygotic deletion model (Cacna1c ± ), including reduced latent inhibition of contextual fear conditioning (Tigaret et al., 2021), impaired appetitive reversal learning (Moon et al., 2018), long-term spatial memory (White et al., 2008), context discrimination in an aversive preparation (Temme et al., 2016), and contextual fear conditioning extinction (Temme and Murphy, 2017).

Learning the relationships between events is critical in the production of adaptive responses, and in changing environments it is vital to determine when a previously learned response is no longer adaptative and, therefore, should no longer be implemented. One example is extinction learning (EL) where, after a response to a cue is acquired on the basis that it predicts aversive or appetitive outcomes, experience of the cue without those outcomes results in the removal of the previously acquired response. This gives an individual the ability to interact flexibly with the environment, a process which is impaired in some psychiatric disorders (Deisseroth et al., 2003; West et al., 2001). While early analyses of EL suggested it was unlearning of the previously learned behavior, more recent evidence suggests that extinction is instead the learning of new relationships (Bouton, 2004). For example, despite successful extinction learning, recover of the response can occur if the individual is re-exposed, either immediately or with a delay in time, to the context in which the original experience was learned (Mendez-Couz et al., 2021; Lengersdorf et al., 2015; Gao et al., 2018; Donoso et al., 2021). This phenomenon is known as renewal (Bouton, 2004; Bouton and Ricker, 1994) and suggests that extinction is not the undoing of prior learning. In addition, extinction studies (Andrianov et al., 2015; André et al., 2015; Mendez-Couz et al., 2021) have implicated neurotransmitter systems and mediators of signaling pathways that are known to be required in other forms of learning (Seyedabadi et al., 2014), as well as the involvement of synaptic plasticity processes believed to underlie new learning (Harley, 2004; Hagena et al., 2016; Lesch and Waider, 2012). It is broadly known that extinction is highly context-specific, thus suggesting the involvement of the hippocampus (Mendez-Couz et al., 2019). The amygdala has also proved to be essential for the extinction of Pavlovian conditioned behaviors (for a review see Bouton et al., 2021). Although most of the work supporting the role of the amygdala, and particularly BLA in EL, comes from fear extinction studies, its role in EL is not limited to aversive conditioning procedures. Previous studies have demonstrated that BLA lesions impair the extinction of appetitive incentive value (Lindgren et al., 2003) and treatment with NMDA antagonists in the avian amygdala impairs the encoding of appetitive EL of context-related conditioned appetitive approach (Gao et al., 2018). Moreover, extinction of an appetitive task involves the absence of a previously present reward, thus suggesting a stimulus-response modulation of the cognitive components of memory systems, for which the amygdala, tightly interconnected with the prefrontal cortex and hippocampus, might play a significant role (McDonald and White, 1993; Ferbinteanu, 2019; Vasquez et al., 2019).

It is noteworthy that most studies of extinction processes, at both behavioral and neural levels, have focused on aversive conditioning (Szapiro et al., 2003; Cammarota et al., 2007; Kim and Richardson, 2009; Ernst et al., 2017). However, it has been demonstrated that appetitive and aversive events are (at least partially) differentially processed (Niyuhire et al., 2007). Moreover, despite the known importance of Cav 1.2 mediated processes in learning and memory more generally, there has been no prior investigation of their potential role in renewal. Therefore, in the present study, we investigated the extinction and renewal of appetitive conditioned magazine approach responses in Cacna1c ± rats. In addition, we used fluorescence in situ hybridization (FISH) to map activity-dependent mRNA expression of Homer1a (H1a) and Arc in the prefrontal cortex and amygdaloid nuclei, following extinction and renewal of the appetitive response. Arc (activity-regulated cytoskeletal-associated protein, also known as Arg 3.1) and H1a are effector IEGs, that have several cellular functions capable of modifying synaptic function (Link et al., 1995; Lyford et al., 1995; Brakeman et al., 1997; Lanahan and Worley, 1998). Their expression can be induced by neural activity (Abraham et al., 1993; Worley et al., 1993) or behavioral stimulation (Sethumadhavan et al., 2020; Hess et al., 1995). Because both genes are activated following novel exploration, Arc and H1a are considered to function together as part of an activity-dependent genomic program to induce and stabilize long-term changes in synaptic efficacy in neural ensembles encoding specific experiences (Vazdarjanova et al., 2002). Due to the brief period of transcription of these genes and the difference in size of their primary transcripts, H1a and Arc have been proven as useful biomarkers for the regional, temporal, and functional differentiation of the contribution of specific brain regions in distinct phases of the learned behavior (Vazdarjanova et al., 2002; Nalloor et al., 2012).