AC5KO mice did not differ from WT littermates in distance traveled when tested in the open field, and showed normal dopamine-dependent locomotor activity (supplemental Fig. environmental stimuli or their own actions with appetitive outcomes is essential for goal-directed behavior and environmental adaptation. Although these forms of appetitive learning, pavlovian and instrumental conditioning, are often integrated, they are dissociable under experimental conditions (Hall, 2002;Kelley, 2004). The distinct neural substrates that underlie these specific forms of appetitive associative learning, however, have not been fully determined and characterized. The cAMP second messenger system is highly conserved and mediates some form of learning in nearly all organisms (Kandel, 2001). Nine membrane-bound isoforms of adenylyl cyclase (AC) are expressed in mammals, each with different expression patterns and regulatory properties (Iwami et al., 1995;Guillou et al., 1999;Hanoune and Defer, 2001). Of these, the calcium/calmodulin (CaCaM)-stimulated AC1 and AC8 XL184 free base (Cabozantinib) have been extensively studied and shown to be critical for hippocampus-based learning and synaptic plasticity because they couple glutamate-mediated increases in intracellular calcium with cAMP production (Wu et al., 1995;Wong et al., 1999;Wang and Storm, 2003). In contrast, the role of AC5 has been less well characterized. AC5 is highly expressed in the striatum, an area strongly associated with reinforcement learning and a major target of dopamine (DA) innervation (Matsuoka et al., 1997). In the striatal regions in which AC5 is expressed, AC1 and AC8 expression is very low (Matsuoka et al., 1997;Cooper et al., 1998;Nicol et al., 2005). The high level of AC5 expression in the striatum suggests that this isoform may be important for certain forms of striatum-dependent learning. Striatal DA has been demonstrated to be critical for both appetitive pavlovian and instrumental conditioning (Schultz et al., 1997;Reynolds et al., 2001;Dickinson and Balleine, 2002;Yin and Knowlton, 2006;Day et al., 2007) and is required for induction of synaptic plasticity at corticostriatal synapses (Calabresi et al., 1992,2007;Wickens et al., 1996;Kreitzer and Malenka, 2007). It has been hypothesized that DA facilitates the learning of environmental contingencies by mediating plasticity mechanisms that strengthen or weaken corticostriatal inputs associated with reward delivery (Wickens et al., 1996,2003;Schultz et al., 1997;Reynolds et al., 2001;Reynolds and Wickens, 2002;Schultz, 2006). AC5 has been shown to be a primary downstream effector of DA receptor signaling, because in mice deficient in AC5 (AC5KO), stimulation of the DA D1or D2receptors does not alter cAMP levels (Iwamoto et al., 2003). In addition, loss of AC5 causes a reduction in D1receptor levels in the striatum (Iwamoto et al., 2003), further suggesting that loss of this isoform may have XL184 free base (Cabozantinib) critical effects on reward learning. Using AC5KO mice, we examined the role of this AC isoform in instrumental and pavlovian conditioning. Although AC5KO mice acquired SETD2 instrumental responding, they exhibited a severe impairment in appetitive pavlovian conditioning and as a result had a difficulty using cues that predicted the availability of reward. This deficit was specific for appetitive conditioning, because aversive conditioning was intact. == Materials and Methods == == == == Mice == ADCY5-deficient (AC5KO) mice were generated as previously described (Iwamoto et al., 2003). AC5KO mice were backcrossed to C57BL/6 for eight generations. Heterozygote offspring were crossed with each other to obtain AC5KO homozygotes and wild-type (WT) controls. All mice tested were 812 weeks of age. All animals were group-housed (four to five per cage) in a temperature- and humidity-controlled barrier facility, with lights on/off at 6:00 A.M./6:00 P.M. All testing was conducted during the light phase. All experiments were approved by the Institutional Animal Care and Use Committee of the University of Chicago. == Behavioral procedures == Appetitive and instrumental conditioning experiments were conducted in mouse operant conditioning chambers that have two retractable levers, a house light, two signal lights above levers, a signal light and a nosepoke hole on the back wall, and a feeder with photobeam (MED Associates). All sessions began with the onset of the house light. == XL184 free base (Cabozantinib) Instrumental conditioning. == In all experiments mice were fedad libitumregular chow in their home cage for 2 h after testing. Naive food-restricted mice were XL184 free base (Cabozantinib) first introduced to the conditioning chambers with two magazine training sessions, in which sucrose pellets were given not-contingently at a variable time of 180 s (VT180), and both levers were retracted. After magazine training, mice were trained on a fixed-interval 20 (FI20) schedule of reinforcement, in which the first lever press after 20 s was reinforced. Sessions ended XL184 free base (Cabozantinib) in 1 h or when mice received 30 rewards. Mice were trained until they reached criterion of.