Effects of D1 receptor knockout on fear and reward learning☆
Introduction
Dopamine neurons generate a wide range of firing responses that have been hypothesized to encode features that guide motivation and learning, such as reward value (Tobler, Fiorillo, & Schultz, 2005), effort (Hamid et al., 2016), and prediction error (Eshel et al., 2016, Schultz and Dickinson, 2000). Phasic dopamine signals, which occur in response to acute stimulation via pharmacological, opto/chemogenetic, or environmental input, appear to be particularly important for learning (Steinberg et al., 2013, Tsai et al., 2009, Waelti et al., 2001). These signals cause a release of dopamine that then binds to different classes of G protein-coupled receptors in dopamine neuron terminal regions. These receptors fall into two broad classes – D1-like dopamine receptors (including D1 and D5) that initiate Gαs or Gαolf signaling and D2-like dopamine receptors (including D2, D3, and D4) that initiate Gαi or Gαo signaling (Beaulieu & Gainetdinov, 2011). Although a great deal is known about how dopamine acts on these receptors, identifying D1-specific effects in learning processes has been difficult because most pharmacological approaches cannot specifically distinguish between D1 and D5 receptors. D1 and D5 receptors are differentially expressed across the brain; thus, specifying the action of one particular receptor subtype would allow for greater precision in therapeutic development.
Pharmacological approaches have found that antagonists and agonists of D1/5 receptors can modulate cocaine-induced conditioned place preference (CPP; Cervo & Samanin, 1995), fear conditioning (Inoue, Izumi, Maki, Muraki, & Koyama, 2000), and fear extinction (Abraham et al., 2016, Hikind and Maroun, 2008). These studies suggest that alterations in phasic signaling through D1-like receptors could have effects on fear and reward processing, but there are currently no pharmacological agents available to distinguish the contributions of D1 and D5 receptors to these behaviors. Thus, genetic approaches are required to examine the specific contribution of D1 receptors to learning (Holmes et al., 2004, Wall et al., 2011).
Previous studies have not shown a consistent effect of D1 receptor knockout (D1 KO) on fear conditioning. For example, El-Ghundi, O’Dowd, and George (2001) demonstrated that expression of contextual fear is unimpaired on the first test day in D1 KO, but that fear remains during repeated extinction tests; that is, D1 KO prevented extinction of contextual fear. In contrast, Ortiz et al. (2010) found impaired cued fear conditioning and Fadok, Dickerson, and Palmiter (2009), found impaired fear-potentiated startle in D1 KO mice. There are procedural variations within these studies that could lead to the different observed outcomes, such as the use of different types of fear conditioning procedures (contextual fear, cued fear, or fear-potentiated startle) that require distinct behavioral responses and circuits. Additionally, increased novelty-induced locomotion in D1 KO mice compared to wildtype (Karlsson, Hefner, Sibley, & Holmes, 2008) could interfere with freezing or potentiated startle measurements. One procedural difference between Ortiz et al., 2010, El-Ghundi et al., 2001 that could decrease expression of fear was the use of one- or two-shock conditioning protocols. When given a one-shock protocol, as in Ortiz et al. (2010), D1 KO mice showed decreased freezing, but with two footshocks, El-Ghundi, O’Dowd, and George (2001) did not observe differences between wildtype or D1 KO mice. Thus, further work is needed to evaluate the effects of D1 KO on learning and memory processes.
In the following experiments, we characterized the behavior of D1 KO mice in fear conditioning and fear extinction to determine whether associative learning can be acquired and maintained without D1 receptors. Based on the studies described above, we tested the effect of both a one-shock and a two-shock cued fear conditioning protocol to determine whether the strength of conditioning impacts the expression of fear in D1 KO mice. The use of cued fear conditioning also allows for an examination of fear responses during CS-off and CS-on periods, which could show whether there are differences in generalized freezing that would impact the expression of fear in D1 KO mice. Additionally, we assess further contextual modulation of extinction by examining renewal of fear after extinction in D1 KO mice. To determine generality of contextual learning effects, we measured cocaine conditioned place preference (CPP) in D1 KO mice (Miner, Drago, Chamberlain, Donovan, & Uhl, 1995). Finally, we used the D1 KO mice to try to isolate whether pharmacological enhancements of extinction induced by a D1/5 agonist (SKF81297; Abraham et al., 2016, Fiorenza et al., 2012, Rey et al., 2014) occur in the absence of the D1 receptor. If they do, it would suggest that D5 receptors are sufficient to mediate these pharmacological effects. We find that contextual and cued fear learning, shock reactivity, and contextual reward learning are retained in D1 KO mice. However, D1 KO mice show decreased contextual fear renewal. We also find that there may be some contribution of D5 receptors to D1/5 agonist-mediated enhancements of fear extinction.
Section snippets
Subjects
Wildtype (male = 19, female = 25), heterozygote (m = 23, f = 22) and D1 receptor knockout mice (m = 11, f = 21) ranging from three to eight months of age were used in these experiments. D1 knockout mice (D1 KO; Drd1aCre/Cre) were generated by insertion of Cre recombinase at the initiation codon of the Drd1a gene locus, resulting in the deletion of the Drd1a gene (Heusner, Beutler, Houser, & Palmiter, 2008). Mice were generated on a 129/Sv background, but backcrossed to C57BL/6 for >10 generations prior to
One trial cued fear conditioning
This experiment examined whether fear learning following a single CS-US pairing is impaired in D1 KO mice compared to WT or D1 HET mice (Ortiz et al., 2010). Due to the small number of knockout mice available for this study, animals were collapsed across sex for statistical power. Fig. 1 shows there were no significant effects of genotype on Acquisition (Acq.), Contextual Extinction (Ctx Ext), Test 1, or Test 2 during CS-on or CS-off periods. This experiment demonstrates that fear learning is
Discussion
There are several key findings in these experiments that help to define the role that D1 receptors may have in learning processes. First, D1 KO mice were unimpaired in cued fear conditioning with either one or two trials or contextual conditioning with one, two, or four trials, suggesting that associative learning may occur in the absence of D1 receptors. Second, this effect was replicated in an appetitive contextual conditioning task, in which D1 KO mice formed a preference for a context
References (35)
- et al.
Effects of dopaminergic and glutamatergic receptor antagonists on the acquisition and expression of cocaine conditioning place preference
Brain Research
(1995) - et al.
Prolonged fear responses in mice lacking dopamine D1 receptor
Brain Research
(2001) - et al.
Modulation of the extinction of two different fear-motivated tasks in three distinct brain areas
Behavioural Brain Research
(2012) - et al.
The neural correlates and role of D1 dopamine receptors in renewal of extinguished alcohol-seeking
Neuroscience
(2007) - et al.
Microinfusion of the D1 receptor antagonist, SCH23390 into the IL but not the BLA impairs consolidation of extinction of auditory fear conditioning
Neurobiology of Learning and Memory
(2008) - et al.
Phenotypic analysis of dopamine receptor knockout mice; recent insights into the functional specificity of dopamine receptor subtypes
Neuropharmacology
(2004) - et al.
Effect of the dopamine D(1/5) antagonist SCH 23390 on the acquisition of conditioned fear
Pharmacology, Biochemistry and Behavior
(2000) - et al.
A parametric analysis of factors affecting acquisition and extinction of contextual fear in C57BL/6 and DBA/2 mice
Behavioural Processes
(2012) - et al.
Effect of cage size on ultradian locomotor rhythms of laboratory mice
Physiology & Behavior
(1997) - et al.
A behavioral genetics approach to understanding D1 receptor involvement in phasic dopamine signaling
Molecular and Cellular Neuroscience
(2011)
Activation of D1/5 dopamine receptors: A common mechanism for enhancing extinction of fear and reward-seeking behaviors
Neuropsychopharmacology
The physiology, signaling, and pharmacology of dopamine receptors
Pharmacological Reviews
Drug-induced conditioned place preference and aversion in mice
Nature Protocols
Dopamine neurons share common response function for reward prediction error
Nature Neuroscience
Dopamine is necessary for cue-dependent fear conditioning
Journal of Neuroscience
Transient neuronal inhibition reveals opposing roles of indirect and direct pathways in sensitization
Nature Neuroscience
High levels of Cre expression in neuronal progenitors cause defects in brain development leading to microencephaly and hydrocephaly
Journal of Neuroscience
Cited by (0)
- ☆
This work was supported by an APA Dissertation Award (ADA), NIH grants DA007262 (ADA), DA018165 (KML), DA025922 (KML), US Department of the Army/DOD-TATRC W81XWH-12-2-0048 (KML), the Merit Review Award BX000810 from the U.S. Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development, Biomedical Laboratory Research and Development (K.A.N.).
- 1
Present address: Department of Pharmacology, University of Washington, United States.