Unresponsive Choline Transporter as a Trait Neuromarker and a Causal Mediator of Bottom-Up Attentional Biases

Abstract

Some rats [sign-trackers (STs)] are prone to attribute incentive salience to reward cues, which can manifest as a propensity to approach and contact pavlovian cues, and for addiction-like behavior. STs also exhibit poor attentional performance, relative to goal-trackers (GTs), which is associated with attenuated acetylcholine (ACh) levels in prefrontal cortex (Paolone et al., 2013). Here, we demonstrate a cellular mechanism, linked to ACh synthesis, that accounts for attenuated cholinergic capacity in STs. First, we found that electrical stimulation of the basal forebrain increased cortical choline transporter (CHT)-mediated choline transport in GTs, paralleled by a redistribution of CHTs to the synaptic plasma membrane. Neither increases in choline uptake nor translocation of CHTs occurred in STs. Second, and consistent with uptake/translocation alterations, STs demonstrated a reduced ability to support cortical ACh release in vivo compared with GTs after reverse-dialysis to elevate extracellular potassium levels. Third, rats were significantly more likely to develop sign-tracking behavior if treated systemically before pavlovian conditioned approach training with the CHT inhibitor VU6001221. Consistent with its proposed mechanisms, administration of VU6001221 attenuated potassium-evoked ACh levels in prefrontal cortex measured with in vivo microdialysis. We propose that loss of CHT-dependent activation of cortical cholinergic activity in STs degrades top-down executive control over behavior, producing a bias for bottom-up or stimulus-driven attention. Such an attentional bias contributes to nonadaptive reward processing and thus identifies a novel mechanism that can support psychopathology, including addiction.SIGNIFICANCE STATEMENT The vulnerability for addiction-like behavior has been associated with psychological traits, such as the propensity to attribute incentive salience to reward cues that is modeled in rats by sign-tracking behavior. Sign-trackers tend to approach and contact cues associated with reward, whereas their counterparts, the goal-trackers, have a preference for approaching the location of the reward. Here, we show that the capacity of presynaptic cholinergic synapses to respond to stimulation by elevating presynaptic choline uptake and releasing acetylcholine is attenuated in sign-trackers. Furthermore, pharmacological inhibition of choline transport induced sign-tracking behavior. Our findings suggest that reduced levels of cholinergic neuromodulation can mediate an attentional bias toward reward-related cues, thereby allowing such cues to exert relatively greater control over behavior.

Keywords: acetylcholine; addiction; attention; choline transporter; motivation; sign-tracking.

Figure 1.

Effects of BF-ES in vivo on CHT-mediated, HC-3-dependent choline transport (n = 20; n = 5 GTs and 5 STs for BF-ES; n = 5 GTs and 5 STs for sham stimulation). After 20 min of BF-ES, cortical and striatal tissues were harvested and synaptosomes were prepared for measuring choline uptake. a, c, Saturation curves for choline uptake by synaptosomes from frontal cortical and striatal tissues. b, d, V_max values for cortical and striatal synaptosomes. In GTs, BF-ES significantly increased the capacity of cortical CHTs to transport choline (b). No such effect was found in STs. Striatal V_max values were unaffected by phenotype or BF-ES. BF-ES may have spread into the striatum to yield an insignificant increase in striatal synaptosomes from GTs (d). K_m values were not affected by phenotype or BF-ES in either region (see Results for ANOVAs; multiple comparisons for this and subsequent figures: *p < 0.05; **p < 0.01; ***p < 0.001).

Figure 2.

Effects of BF-ES on subcellular CHT distribution in cortical synaptosomes (n = 19; n = 5 GTs and 5 STs for BF-ES; n = 4 GTs and 5 STs for sham stimulation; the immunoblots depict duplicates from 1 ST and 1 GT). a, Consistent with previous research, at baseline (sham stimulation), in GTs and STs, the majority of CHTs was located on intracellular domains, such as vesicular and endosomal membranes. BF-ES lowered the intracellular density of CHTs in both phenotypes [main effect, no interaction with phenotype; sham, 65.28 ± 12.82 (normalized density); BF-ES, 45.77 ± 9.86]. Moreover, STs generally exhibited lower levels of intracellular CHTs than GTs (main effect of phenotype). b, Whereas BF-ES increased the density of CHTs in the plasma membrane-enriched LP1 fraction in GTs, this was not the case in STs. Results from additional analyses are illustrated in c. In synaptosomes from sham-stimulated animals, the LP2/LP1 ratio was already near 1 (indicated by red protein symbols in membrane). Previous findings indicated that a ratio of 1 represents the limit of CHT mobilization to plasma membrane. Thus, synaptosomal CHT distribution in STs may have already reached this maximum at baseline, primarily attributable to a lower intracellular CHT density than in GTs. Second, in STs, the BF-ES-induced loss of LP2 CHTs (blue protein symbols) was not reciprocated with an increase in LP1 CHTs, suggesting stimulation-induced translocation of a portion of intracellular CHTs to domains other than synaptic plasma membrane and those captured in the present LP2 fraction.

Figure 3.

Effects of reverse-dialysis of potassium (100 mm) on cortical levels of ACh, adenosine, and GABA (see Table 1 for a summary of effects on all neurotransmitters and metabolites that were determined in this experiment; n = 11; n = 6 GTs and 5 STs). K⁺ was perfused for 15 min (five 3 min collections). a, b, Significant interactions between the effects of phenotype and stimulation were found only for ACh (a) and adenosine (b; see Table 1 for ANOVAs). Because of the variability of absolute ACh levels, post hoc comparisons did not locate the interaction (see Results for additional analyses of data expressed as percent change from baseline). Adenosine levels were less variable and were significantly higher in GTs for the first three collections during stimulation. c, GABA levels increased similarly in GTs and STs (see Table 1 for other analytes that reached comparable levels during stimulation in the two phenotypes).

Figure 4.

Effects of the CHT inhibitor VU600122 on pavlovian conditioned approach behavior toward a lever (CS) versus the location of food delivery (food cup; n = 49; n = 25 treated with VU600122; n = 24 treated with vehicle). Behavior was measured over five training sessions to determine lever- and food cup-directed behavior after administration. a, Drug or vehicle was given before sessions 2–5. Data from session 1, and after the administration of vehicle, were used to exclude rats that exhibited an early trend for sign-tracking behavior, to allow for a more “aggressive” test of the hypothesis that VU600122 fosters sign-tracking. b–f, The plots depict mean ± SEM for food cup or magazine (mgz.) contacts evoked by the CS (b), number of lever contacts (c), number of non-CS food magazine entries (d), latency to first lever contact after CS presentation (e), and latency to the first food magazine entry after CS presentation (f). Compared with the effects of vehicle, administration of VU600122 facilitated lever-oriented behavior and reduced CS-evoked magazine-oriented behavior (for main effects of treatment and interactions, see Results; multiple comparisons indicated in c and f were based on significant interactions between the effects of VU600122 and session; see Results).

Figure 5.

Individual PCA scores for rats treated with vehicle (a) or VU600122 (b) (final numbers per phenotype and treatment are indicated on the right of the two graphs). Note that session 1 scores were obtained in the absence of treatment and that rats with PCA scores >0, and thus showing an early trend for sign-tracking, were excluded from this experiment to bias it against the expected effect of the drug. VU600122 or vehicle was given before sessions 2–5. Individual rats' PCA scores from sessions 4 and 5 were averaged to assign the final phenotype. Compared with vehicle, treatment with VU600122 significantly increased the proportion of STs after the final session 5.

Figure 6.

Attenuation of potassium-evoked increases in cortical extracellular ACh levels by VU600122 (n = 4 GTs). VU600122 was administered at the same dose (0.3 mg/kg) used to demonstrate the manifestation of sign-tracking behavior. VU600122 significantly attenuated potassium-induced increases in ACh levels (main effect of drug; see Results).