Prefrontal cholinergic mechanisms instigating shifts from monitoring for cues to cue-guided performance: converging electrochemical and fMRI evidence from rats and humans

Abstract

We previously reported involvement of right prefrontal cholinergic activity in veridical signal detection. Here, we first recorded real-time acetylcholine release in prefrontal cortex (PFC) during specific trial sequences in rats performing a task requiring signal detection as well as rejection of nonsignal events. Cholinergic release events recorded with subsecond resolution ("transients") were observed only during signal-hit trials, not during signal-miss trials or nonsignal events. Moreover, cholinergic transients were not observed for consecutive hits; instead they were limited to signal-hit trials that were preceded by factual or perceived nonsignal events ("incongruent hits"). This finding suggests that these transients mediate shifts from a state of perceptual attention, or monitoring for cues, to cue-evoked activation of response rules and the generation of a cue-directed response. Next, to determine the translational significance of the cognitive operations supporting incongruent hits we used a version of the task previously validated for use in research in humans and blood oxygenation level-dependent (BOLD)-functional magnetic resonance imaging. Incongruent hits activated a region in the right rostral PFC (Brodmann area 10). Furthermore, greater prefrontal activation was correlated with faster response times for incongruent hits. Finally, we measured tissue oxygen in rats, as a proxy for BOLD, and found prefrontal increases in oxygen levels solely during incongruent hits. These cross-species studies link a cholinergic response to a prefrontal BOLD activation and indicate that these interrelated mechanisms mediate the integration of external cues with internal representations to initiate and guide behavior.

Figure 1.

Schematic illustration of the SAT (rat version), main sequences of trials and definition of terms, and schematic overview of the presence or absence of cholinergic, BOLD-fMRI and tissue oxygen responses. The schematic foreshadowing of results assists in maintaining the overview of findings from diverse methods and two species. a, The SAT consists of randomized sequences of signal and nonsignal trials (signal durations were 500, 50, and 25 ms for rats and 60, 39, and 25 ms for humans). Following a signal or nonsignal event, rats were prompted to respond by extending both levers (data shown) while an auditory cue served to open the response window for humans. Correct responses were hits and correct rejections for signal and nonsignal trials, respectively, and in rats were rewarded with water and in humans with a cue indicating monetary reward. Incorrect responses were misses and false alarms, respectively, and triggered the next ITI (see Materials and Methods for more details). b–g, Overview over key trial pairs resulting in the presence or absence of neuronal responses during the second trial. All three measures of neuronal activity exhibited increases in signal-hit trials that were preceded by correct rejections or misses (incongruent hits) but not by prior hits (consecutive hits; b–d). Correct rejections never generated increases in neuronal responses, regardless of whether they were incongruent with the prior trial or part of a chain of consecutive correct rejections (e, f). Likewise, misses did not evoke neuronal activity regardless of preceding trial type (g). Insufficient data from trial sequences involving false alarms prohibited the analysis of such sequences.

Figure 2.

Illustration of electrodes, measurement scheme, and cholinergic transients during incongruent hits. a, Illustration of the four Pt recording sites fabricated onto ceramic bases and the approximate placement of the recording sites in the prelimbic (Prl) cortex. CO was immobilized onto 2/4 Pt sites and all sites were equipped with a Nafion layer to repel electro-active interferents ascorbic acid, uric acid, and dihydroxyphenylacetic acid (DOPAC). ACh is hydrolyzed by endogenous acetylcholinesterase (AChE) and the resulting choline is oxidized by immobilized CO on the electrode (Giuliano et al., 2008). The resulting hydrogen peroxide is then detected amperometrically. Current from sites not equipped with CO was used for self-referencing (see Materials and Methods for details and in vitro calibration). b, Choline concentrations (M; SEM) recorded during signal-hit trials as a function of prior trial type (Fig. 1b–d for illustration of consecutive and incongruent hits). The time of signal presentation (t = 0 s) and lever extension (2 s) are indicated on the bottom. ANOVA (see Results) indicated that only incongruent hits were associated with increases in cholinergic activity (n = 5, based on 122 incongruent and 69 consecutive hit trials). Furthermore, compared with baseline cholinergic activity (2 s before the signal), cholinergic activity was significantly increased by the time the levers were extended and thereafter, peaking 6.5 s after the signal (red data points). c, Choline concentrations during consecutive and incongruent correct rejections (n = 5, based on 90 incongruent and 146 consecutive correct rejections; Fig. 1e,f). Response latencies did not differ between consecutive and incongruent correct rejections and were 640.51 ± 34.57 ms after the extension of the levers (F_(1,212.99) = 3.35, p = 0.07). Recordings did not differ between the two conditions. Comparisons against baseline choline concentrations indicated that there was an insignificant trend for a decline in current during incongruent correct rejections, peaking 1 s following lever extension (see Results).

Figure 3.

The contrast between incongruent and consecutive hits activated right rostrolateral PFC (BA 10; shown in a on CARET slightly inflated surface representation, and on slices using the MRIcron high-resolution template; n = 15). Thalamic activations are partially displayed (see Table 1 for all activations meeting the combined height threshold of p < 0.0001 and cluster volume threshold of 20 voxels). b, Linking human imaging results to the cholinergic system, at a slightly reduced threshold right basal forebrain also showed significant activation for the contrast between incongruent and consecutive hits. c, Response times were shorter for incongruent hits than consecutive hits, and BA 10 activation differences between incongruent and consecutive hits correlated with reaction time differences (r = −0.58; p < 0.05). Participants with the greatest increase in prefrontal activation showed the greatest response time benefit for incongruent hits.

Figure 4.

The contrast between incongruent and consecutive hits revealed significant functional connectivity between the right rostrolateral PFC seed region (BA 10; shown in green; t > 5) and parietal cortex regions associated with attention (superior parietal lobule, BA 7; warm colors), as well as middle frontal gyrus (BA 10/46). The functional connectivity t map is displayed at a p < 0.0001 height, 100 voxel cluster threshold to highlight those regions showing strongest connectivity; see Table 3 for a full list of regions meeting our a priori threshold. Evidence of enhanced functional connectivity with parietal regions associated with controlled attention and the integration of stimuli with responses (Corbetta et al., 1993, 2008; Stephan et al., 1995; Hawkins et al., 2013) strengthens the hypothesis that rostrolateral PFC activity supports shifts from monitoring to cue-directed behavior.

Figure 5.

Amperometric measurements of prefrontal oxygen levels during incongruent hits. a, illustrates the placement of Teflon-insulated silver electrodes and the carbon tip in the thalamic input layer of the prelimbic cortex. b, Currents recorded before the onset of the task and during the entire task session, indicating a continuous decline in current across session (see Results for ANOVA). Note that the ordinate in b is in nA while those in c and d are in pA. c, During incongruent hits, when compared with consecutive hits, peak oxygen levels were significantly higher (n = 9, based on 331 incongruent and 127 consecutive hit trials). Moreover, incongruent hit-associated levels increased significantly from baseline levels, with post hoc comparisons indicating significantly elevated T_O2 levels present beginning for 0.5 s following the signal (red data points). T_O2 levels declined for all other trial types, reflecting the session-based trend for decreasing T_O2 levels.