Task Acquisition and Performance during Recording Sessions
Animals reached criterion performance for each stage of learning of the cued appetitive response task within about 2 weeks of training. The latencies between cue presentation and reward retrieval decreased continuously during the two stages of task acquisition (), as indicated by a significant effect of day (stage 1, 10-s cue followed by immediate reward: F(4,20)=27.25, p<0.001; stage 2: 1-s cue followed by reward 6±2s later: F(4,20)=8.98, p<0.001).
In sessions during which cholinergic activity was recorded, animals detected significantly more cues than they missed (58.7±2.3 % of the cues were detected; 25 trials/session; t(10)=5.03, p<0.001; ). As would be expected, the latencies between reward delivery and reward retrieval were longer in trials in which cues were missed (t(10)=2.26, p=0.048; ).
Detected Cue-Evoked Transient Increases in Cholinergic Activity
Cue-Evoked Transient Increases in Cholinergic Activity in the mPFC
Details concerning electrode preparation, in vitro
calibration and electrode properties in vivo
following completion of the recording experiments are described in Supplemental Materials
. Amperometric recordings of cholinergic activity in the mPFC, but not motor cortex (Supplemental Materials
), revealed transient increases that were evoked by cues that were detected (). Cue-evoked cholinergic signal amplitudes were significantly higher for detected cues when compared with missed cues (highest choline signal levels observed during the 6±2s cue-reward interval; t(10)
= 4.21, p=0.002; ). The time required for cholinergic signal amplitudes to decrease by 50% from peak (t50
) was 3.17±0.27 s. As will be further substantiated below, during trials involving missed cues, cholinergic activity remained unchanged from pre-cue levels ().
Additional analysis indicated that reward delivery and retrieval did not evoke cholinergic activity. First, choline signal levels recorded for 2 s prior to and 5 s following reward delivery did not differ by trial type (detected/missed; t(10)
=1.18, p=0.27). Second, in trials involving missed cues, choline signal levels recorded for 5 s following the (missed) cue and following reward delivery did not differ (t(10)
=2.17, p=0.10; ). The conclusion that reward-related processes did not confound cholinergic activity is further supported by the demonstration of regular cue-evoked cholinergic transients in catch trials not involving reward delivery, and by the absence of such transients early into the acquisition of the task (for these results see Supplemental Materials
As the definition of detection involves the initiation of a behavioral response that indicates the entrance of a behaviorally significant cue into the processing stream (Introduction), the onset of the cue-evoked behavioral response was expected to correlate with the onset of the increase in cholinergic activity. Such increase in cholinergic activity was defined as the time point, relative to cue presentation, when cholinergic activity increased by 25% over pre-cue levels. As illustrated in , the time of onset of the choline spike correlated significantly with the onset of the behavioral shift (Pearson’s r=0.79, p<0.001).
In this task, the efficacy of the cue detection process is indicated by response latencies. Choline signal amplitudes correlated significantly with the latencies between cue presentation and reward retrieval (Pearson’s r=−0.37, p=0.045). Analysis of the regression between these two variables indicated that an increase in choline signal amplitude by 1 μM was associated with a decrease of 1.75 s in response latency.
Left-Shift of Cue-Evoked Cholinergic Signals
The evidence described above was based on recordings in the mPFC of rats performing the cued appetitive response task involving a 6±2 s interval between cue and reward delivery (). Cholinergic activity was recorded in a separate group of animals trained to perform the cued appetitive response task involving a shorter (2±1 s; ) interval, in order to test the following hypothesis. If cue-evoked cholinergic transients merely reflect the sensory encoding of the cue, the timing of cue-evoked cholinergic activity should be insensitive to variation of the interval between cue and reward delivery. In contrast, if variation of this interval causes variation of the timing of the cue-evoked cholinergic transients, such a finding would indicate that cholinergic transients reflect a shift in the timing of cue-evoked cognitive operations that collectively define detection (Introduction). As illustrated in , the latency from cue presentation to the (detected) cue-evoked choline signal peak amplitude was significantly shorter in animals performing the task involving the shorter cue-reward interval (t(53)=9.26, p<0.001; ). The amplitudes of the cholinergic transients did not differ between the two task versions (t(9)=1.72, p>0.12). As was the case for recordings from the mPFC of animals performing the task involving the longer cue-reward interval, cholinergic activity evoked by detected cues was significantly higher when compared with missed cues (t(8)=6.97, p<0.001). Cholinergic activity in trials involving missed cues and reward delivery-evoked port approach remained at pre-trial levels (; see below for statistical results).
Effects of a Shorter Cue-Reward Interval on the Timing of Cholinergic Transients
Based on the choline signal population data for detected trials from both task versions, over the entire 16 s period (see and ), the effects of the variation of the cue-reward interval were indicated by a significant interaction between the effects of time (data across 16 s) and cue-reward interval (long, short) on choline signal levels (main effect of time: F(1,31)
=13.28, p<0.001; main effect of interval: F(1,53)
=21.38, p<0.001; time × interval: F(31,1643)
=10.72, p<0.001). In the analysis of choline signal levels recorded during trials in which the cue was missed, neither an effect of time or interval nor an interaction between these two factors were found (both p>0.05), reflecting the absence of changes in cholinergic activity (, ). Cue-evoked cholinergic transients were not observed in separate experiments in which cholinergic activity was recorded in the motor cortex (Supplemental Materials
Pre-Cue Trends on Cholinergic Activity
In the analysis of cholinergic signal levels across trials involving cue detection and misses, respectively, systematic relationships between pre-cue trends in cholinergic signal levels in the mPFC and trial outcome (detection or miss) were discovered. For a systematic analysis of this relationship, data from a 20-s period prior to the cue was boxcar-filtered and the slope of the linear regression was determined (see Supplemental Methods
). As illustrated in , in 80% of trials involving cue detection, mPFC pre-cue cholinergic activity showed a negative trend; conversely, 83% of misses were preceded by increases in cholinergic activity (χ2
=24.15, p<0.001). Moreover, for trials with detected cues, steeper decreases in pre-cue cholinergic activity correlated with greater amplitudes of cue-evoked cholinergic activity (Pearson’s r=−0.32, p=0.01).
Pre-Cue Trends in Cholinergic Activity Predict Trial Outcome
A similar result was found in the analysis of cholinergic activity recorded in the motor cortex (76% and 72%, respectively; χ2=9.70, p=0.002; ). The magnitude of these trends did not differ between mPFC and motor cortex (; decreases preceding cue detection: t(41)=0.038, p=0.97; increases preceding misses: t(41)=0.93, p = 0.36).
Cholinergic Deafferentation of the Recording Area Abolishes Cue-evoked Cholinergic Transients
In order to confirm that the demonstration of evoked cholinergic activity, measured by choline-sensitive microelectrodes, requires the presence of cholinergic terminals, cholinergic activity was recorded following the unilateral removal of cholinergic inputs to the recording region (see Methods). In contrast to bilateral cholinergic deafferentation of the mPFC (below), such restricted deafferentation is insufficient to impair attentional performance (Gill et al., 2000
) and, likewise, did not affect the proportion of cues that was detected (t(9)
=1.75, p=0.22). Detected cue-evoked cholinergic activity was not observed in the deafferented recording region, confirming the validity of the measure in terms of reflecting ACh released from cholinergic neurons ().
Cholinergic Deafferentation-Induced Attenuation of Cue-Evoked Cholinergic Signals and Cue Detection
Bilateral Cholinergic Deafferentation-Induced Disruption of Cue Detection
Bilateral removal of mPFC cholinergic inputs decreased the proportion of detected cues (F(3,16)
=8.68, p=0.001; ). Multiple comparisons indicated that this impairment was present during all three weeks of post-surgery training and testing (all p<0.025). The number of port approaches was recorded across test sessions (see Methods), regardless of whether such approaches were evoked by cue or reward delivery. The effects of the lesions on this measure were analyzed in order to reveal potential confounds based on general exploratory or activity changes. Although the lesion produced a significant effect on this measure (F(3,16)
=3.46, p=0.041), multiple comparisons indicated that this was due to an increased frequency of port approaches observed during the second week after the infusions of the immunotoxin (). Immunotoxin-induced deafferentation typically reaches asymptotic levels two weeks post-injection (Waite et al., 1994
In contrast to the effects of bilateral cholinergic deafferentation of the mPFC, a similar deafferentation of the motor cortex did not affect cue detection rate (F(3,16)
=0.55, p=0.67; see Supplemental Materials
Minute-Based, Performance Session-Associated Changes in Cholinergic Activity in mPFC and Motor Cortex
The transient increases in cholinergic activity that were recorded in the mPFC during trials involving detected cues were superimposed over more slowly changing (on the scale of minutes), or tonic, changes in cholinergic activity. Such tonic cholinergic activity was also observed in the motor cortex (). ANOVA confirmed that session-related changes in cholinergic activity occurred in both cortical regions (main effect of time: F(39,351)=2.13, p<0.001) and they did not differ in magnitude (main effect of region: F(1,9)= 0.32, p=0.59).
Tonic Changes in Cholinergic Activity
Performance-associated increases in mPFC tonic cholinergic signal levels were positively correlated with the amplitudes of cue-evoked cholinergic transients (Pearson’s r=7.21, p<0.001; ) and with a greater proportion of detected cues (analyzed over blocks of 5 trials each; r=0.46, p=0.01). Tonic signal levels recorded in the motor cortex were not correlated with performance (r=0.04, p=0.86). Furthermore, the total number of port approaches, a measure of task-related locomotor and exploratory activity, did not correlate with tonic levels of cholinergic activity recorded in mPFC or motor cortex (both p>0.05). Session-related tonic cholinergic activity corresponded with levels of ACh release measured by using microdialysis in both cortical regions (Supplemental Materials
In animals trained to perform the task and placed into the test chamber without activating the task, no such tonic changes in cholinergic activity were observed, indicating that performance of the task is necessary to evoke such tonic changes, and that context alone and expectation of performance were not sufficient in evoking tonic increases in cholinergic activity (mPFC: F(5,17)=0.49, p=0.78; motor cortex: F(5,17)=0.83, p=0.55; ). Finally, session-related tonic changes in mPFC cholinergic activity were not observed following unilateral removal of cholinergic inputs to the recording region (F(5,29)=0.77, p=0.58).