Relation of locus coeruleus neurons in monkeys to Pavlovian and operant behaviors

Abstract

Noradrenaline is released throughout the forebrain from locus coeruleus (LC) projections in close temporal proximity to emotional and goal-directed events. To examine interactive influences of these processes on LC neuronal activity, we used a task where Pavlovian and operant processes vary and can be easily identified. We recorded 69 single LC neurons from two monkeys performing a task where cues indicate the progression through schedules of one, two, or three operant trials. Pavlovian responses and phasic LC activations occur following the appearance of conditioned visual cues (54/69 neurons), especially those at the beginning of new schedules, whether the current trial will be rewarded (single trial schedule) or not (2 or 3 trial schedules), and after visual imperative signals eliciting the operant response (64/69 neurons), whether the current trial will be rewarded or not. The modulation of LC responses seems to be relatively independent of attention or motivation, because the responses do not covary with operant performance in the task. The magnitude of LC responses across the schedules varied in close relation to the intensity of Pavlovian behavior but these responses were also modulated by operant processes. Our conclusion is that LC activation occurs when task-relevant stimuli evoke a conditioned instinctive (Pavlovian) response, with the strength of the activation also being modulated by goal-directed processes. Thus locus coeruleus neurons broadcast information about stimulus-elicited primitive and goal-directed behaviors to forebrain structures important for executive functions and emotions.

FIG. 1.

Behavioral tasks. A: sequential color-discrimination trial. A trial was initiated when the monkey touched a bar. A cue appeared (black bar). Colored dots, red (Wait), green (Go), and blue (OK, after correct bar release), appeared in sequence. B: trial sequences in the reward schedule task. The rectangular cue's brightness changed with the schedule state, in proportion to the schedule fraction. A black cue was always used when the trial was to be rewarded. The brightness of the cue changed at the beginning of each trial. To move on to the next schedule state, the monkey had to complete the current trial. After an incorrect trial, the same cue was repeated until the trial was performed correctly. C: Pavlovian schedule task and cues. In the Pavlovian schedule task, cues also indicated how many trials had to be completed before obtaining the reward. In contrast to the operant task, there was no color discrimination or operant response; the blue dot appeared 2.5 s after cue onset.

FIG. 2.

Recording localization and pharmacological characterization. A: MR image (1.5 T) of monkey D with an electrode at 1 site where locus coeruleus (LC) neurons were identified by physiological criteria. Structures are labeled on the side contralateral to the recordings to avoid obscuring the electrode track. The electrode goes through the inferior colliculus (IC) and terminates in the region known to contain the LC, lateral from the 4th ventricule (IV). B: brain section from the same monkey, stained for tyrosine hydroxylase (1.25×). The LC appears in black laterally from the 4th ventricle (labeled IV). Tracks directed toward the LC are visible in the IC. Arowheads point to electrolytically made microlesions in the LC. C: brain section stained with thionine (5×), 3 sections anterior form the one shown in B. Arrowheads indicate the microlesions shown in B. An electrode track (arrow) can be seen 1 mm lateral from the microlesions. D: pharmacological test of a presumptive LC neuron. Clonidine (20 μg/kg, ip) was injected at the time represented by the labeled arrow. Following the brief injection-induced activation of the cell, there was a long-lasting inhibition that disappears over 10s of minutes.

FIG. 3.

Operant behavior in the reward schedule task. A: error rates for the 6 schedule states (fractions on x-axis) in valid cue (black solid line, filled symbols) and random cue (gray broken line, open symbols) conditions. The data were averaged over all the sessions for monkey K (circles) and monkey D (squares). The error rates decreased as the number of trials remaining before the reward decreased in the valid cue condition. In the random cue condition, the schedules were still in effect, but the cue displayed in any trial was chosen randomly. Both the factors of “schedule state” and “condition” were significant, and there was a significant interaction [2-way ANOVA, monkey D: F(5) = 5.1, P = 5 × 10⁻⁸, r² = 12% and F(1) = 6.9, P = 0.009, r² = 1.2% for schedule state and condition, respectively, and F(5,456) = 9.1, P = 3 × 10⁻⁸, r² = 7.9%, for the interaction; monkey K: F(5) = 6.2, P = 1.4 × 10⁻⁵, r² = 5.6% and F(1) = 12.8, P = 0.0004, r² = 2.3%, F(5,486) = 5.1, P = 0.0001, r² = 4.6%]. In the random cue condition, the error rates were indistinguishable across schedule states. In the valid cue condition, there were significantly fewer errors in rewarded trials than in unrewarded trials (Tukey test, P < 0.01). B: reaction times of the operant response in the 6 schedule states. In the valid cue condition, the reaction times get shorter as monkeys approach the reward. In the random cue condition, reaction times were indistinguishable across schedule states. There was a significant effect of schedule state, condition, and a significant interaction between the 2 factors [monkey D: F(5) = 14.5, P = 3 × 10⁻¹³, r² = 12% and F(1) = 8, P = 0.005, r² = 1.3% for schedule state and condition, respectively, and F(5,456) = 11.8, P = 1 × 10⁻¹⁰, r² = 9.9%, for the interaction; monkey K: F(5) = 3, P = 0.01, r² = 2.8% and F(1) = 14.8, P = 0.0001, r² = 2.8%, F(5,486) = 2.5, P = 0.03, r² = 2.3%].

FIG. 4.

Example of lipping behavior in the reward schedule task. A: lipping signal around cue onset in the 6 schedule states, indicated by corresponding fraction and cue for a representative session in the valid cue condition. Each line is the signal (in mV, for 1 trial) from the strain gauge attached to the sipper tube positioned between the monkey's lips. On the y-axis, traces are offset by 250 mV for clarity of viewing. Traces are aligned on cue onset (black vertical line, t = 0) for each trial, sorted in increasing latencies between cue onset and onset of the blue fixation spot (gray dot). Time around cue onset is in seconds. The monkey showed a strong, reliable lipping response after each 1st cues of a schedule, irrespectively of cue brightness. B: lipping signal around bar release (t = 0, vertical line). Gray dots indicate trial outcome (cue off, with or without reward delivery). Other display conventions as in A. In 1st states (1/1, 1/2, and 1/3), the cue-elicited response is visible before bar release. The monkey displays a small but significant lipping response at the time of the bar release. This response is more pronounced in rewarded trials. Sustained (unconditioned) lipping occurs after reward delivery in 1/1, 2/2, and 3/3 states. C: lipping signal around correct (left) and erroneous (right) bar release (t = 0, vertical line). In this session, the monkey made numerous erroneous bar releases, which, in contrast to correct bar releases, are not associated with lipping responses.

FIG. 5.

Pavlovian responses in the reward schedule task. A: percentage of trials with lipping responses to cues (left) and bar release (right). The percentage of trials with lipping responses to cues is higher in first trials [in valid cue condition, monkey D: 46 vs. 25% χ²(1) = 179, P < 1 × 10⁻¹⁰; monkey K: 76 vs. 65%, χ²(1) = 70, P < 1 × 10⁻¹⁰; in random cue: monkey D: 19 vs. 11% χ²(1) = 87, P < 1 × 10⁻¹⁰; monkey K: 43 vs. 34%, χ²(1) = 57, P < 1 × 10⁻¹⁰]. At bar release, the proportion of lipping responses is significantly higher in rewarded than in nonrewarded trials [monkey D: 32 vs. 25%, χ²(1) = 22, P = 5 × 10⁻⁶; monkey K: 78 vs. 74%, χ²(1) = 12, P = 0.0006]. For both cue onset and bar release, more trials had lipping in the valid cue than in the random cue condition [cues: monkey D: 36 vs. 15%, χ²(1) = 621, P < 1 × 10⁻¹⁰; monkey K: 71 vs. 39%, χ²(1) = 1,123, P < 1 × 10⁻¹⁰; bar release: monkey D: 29 vs. 13%, χ²(1) = 418, P < 1 × 10⁻¹⁰; monkey K: 76 vs. 46%, χ²(1) = 1,054, P < 1 × 10⁻¹⁰]. B: latency of lipping responses. At cue onset, lipping latencies were shortest in 1st trials (504 and 208 ms for monkeys D and K, respectively) and longer in subsequent trials [628 and 408 ms, t(979) = 5, P = 8 †× 10⁻⁸ and t(2,359) = 17, P < 1 × 10⁻¹⁰ for monkeys D and K, respectively). Latencies of response to bar release were shorter (134 ± 3 and 120 ± 1 ms for monkeys D and K, respectively) and indistinguishable across schedule states. C: lipping responses to cues in the Pavlovian schedule task. As in the reward schedule task, more trials had lipping in the valid cue than in the random cue condition [monkey D: 22 vs. 7%, χ²(1) = 180, P < 1 × 10⁻¹⁰; monkey K: 65 vs. 53%, χ²(1) = 31, P = 2.4 × 10⁻⁸]. The percentage of trials with lipping responses to cues is higher in 1st trials [in valid cue condition: monkey D: 35 vs. 10%, χ²(1) = 118, P < 1 × 10⁻¹⁰; monkey K: 69 vs. 62%, χ²(1) = 8, P = 0.005].

FIG. 6.

Activity of an LC unit. Raster and overlaid spike density displays of neural activity. Each row of dots shows spike times in 1 trial. The continuous gray line shows the spike density, representing the average firing per millisecond per trial over all trials. Left: aligned on cue onset (t = 0, black vertical line). Trials are displayed in chronological order (1st at top). The line of open circles at t = 500 ms indicates the time when the red fixation point appeared. Right: activity aligned on operant response (bar release). The open squares show the time when the green spot (go signal) appears. Trials are sorted by reaction time (longest on top).

FIG. 7.

Magnitude and schedule sensitivity of LC responses. A: mean response magnitude of LC neurons showing a significant response to cues (left) and bar release (right) in the reward sxchedule task. Error bars indicate SE. Response magnitude is the firing rate (in spikes/s) during response windows (500 ms after cue onset or 250 ms before bar release). Responses were stronger at bar release than at cue onset, with no difference between valid cue and random cue conditions [2-way ANOVA, effect of stimulus: F(1) = 23.3, P = 3.0 × 10⁻⁶, r² = 12%; effect of condition: F(1) = 0.005, P = 0.09, r² = 0.2%; interaction: F(1,175) = 0.02, P = 0.96, r² = 0%]. B: the sensitivity to schedule states was quantified for each cell by measuring the variance accounted for by a 6-level schedule state factor on response magnitude using an ANOVA. Mean and SE across the population of responding neurons are shown. For both responses to cues (left) and bar release (right), responses are more selective to state in the valid cue than in the random cue condition. [2-way ANOVA, significant effect of stimulus: F(1) = 11.5, P = 0.008, r² = 5.9%; significant effect of condition: F(1) = 7.6, P = 0.006, r² = 3.9% and no significant interaction: F(1,175) = 0.4, P = 0.5, r² = 0.2%].

FIG. 8.

Responses to cues, example of cell showing a 1st–non-1st effect. Activity of an LC neuron around cue onset in the valid cue condition of the reward schedule task. Plotting conventions as in Fig. 6. Responses are shown in the 6 schedule states, indicated by fractions. Inset: mean and SE of responses across the 6 schedule states. The response was significantly stronger in trials indicating the beginning of a schedule (1/1, 1/2, and 1/3) than in subsequent trials. Such response patterns were classified as 1st–non-1st effect.

FIG. 9.

Average standardized responses of neurons showing 1st–non-1st and state responses to cues. Selective neurons were classified as 1st–non-1st, reward–no-reward, or state as a function of their response pattern across schedule states. A: mean standardized responses (z scores) across schedule states for the 18 neurons classified as 1st for their response to cues. Gray levels were attributed randomly to each neuron to facilitate viewing. Responses of the 3 cells for which response magnitudes were higher in non-1st than in 1st states were multiplied by −1 for graphical purposes. For neurons classified as 1st–non-1st, the amount of variance explained by the 6-level schedule state factor in a 1-way ANOVA was not significantly different form the variance explained by the 2-level (1st–non-1st) factor in another ANOVA. B: mean standardized responses of the 15 neurons classified as state for their response to cues. The responses show an idiosyncratic pattern, and the amount of variance explained by the 6-level ANOVA was significantly higher than the variance explained by either of the 1-level ANOVAs for 1st–non-1st and reward–no-reward effects.

FIG. 10.

Responses to cues, example of cell showing a state effect. Activity of an LC neuron around cue onset in the valid cue condition of the reward schedule task. Responses are stronger in 1st trials but, in addition, the activation is significantly stronger for the cue indicating the 1 trial schedule (which is both 1st and rewarded). The response to the cue indicating the 2nd trial of 3 trials schedules, which is neither 1st nor rewarded, is the weakest. The 6-level state factor accounts for significantly more variance than either 1st or reward factors. This cell was classified as displaying a state effect.

FIG. 11.

Categories of LC responses. Percentage of the different categories of LC neurons in valid (n = 69 neurons) and random cue conditions (n = 48 neurons) for responses to cues and before bar release. For each bar, the proportion of unresponsive neurons is shown in white. Responding neurons were broken down into nondiscriminative neurons (light gray) and neurons presenting a significant discrimination across the 6 schedule states (6-level ANOVA). For responses to cues, the proportion of responding neurons was significantly higher in valid cue than in random cue conditions. In the valid cue condition, most responding neurons discriminated across schedule states, with an equivalent proportion of neurons classified as 1st–non-1st and state. In the random cue condition, the proportion of selective neurons was smaller than in the valid cue condition, and virtually all of them were classified as 1st. Most LC neurons responded before the operant response, with no significant difference between the proportion of responding neurons in valid cue and random cue conditions [χ²(1) = 1.7, P = 0.2]. Compared with cue responses, only a small proportion of neurons responding before bar release discriminated across the 6 schedule states and about one half of the selective bar-release neurons showed a reward–no-reward effect in the valid cue condition.

FIG. 12.

Population responses of LC neurons. A: in the reward schedule task, at cue onset, the population response is stronger in 1st trials vs. subsequent trials. In the valid cue condition, the population response also shows a state effect. In the random cue condition, neurons only distinguish between 1st and non-1st cues (1st effect). B: at bar release, differences across schedule states were smaller. In the valid cue condition, the response magnitude of the population was significantly weaker in rewarded trials (1/1, 2/2, and 3/3) compared with nonrewarded trials (1/2, 2/3, and 3/3). In the random cue condition, responses were weaker in 1st compared with non-1st trials. C: population responses to cues in the Pavlovian schedule task. Although the variance in this task is larger than in the reward schedule task (fewer neurons were recorded), these cells also displayed significantly stronger responses to cues in 1st compared with non-1st cues. This 1st effect was significant in both valid and random cue conditions.

FIG. 13.

Correlation between LC and lipping responses. The magnitude of lipping and LC responses in each trial was standardized using a z-score procedure within each session, and we tested the strength of the correlation between these 2 variables. For cue-related activity (left), there was a significant positive correlation between the magnitude of neuronal and lipping responses [black line, slope = 0.07, adjusted r² = 0.07, F(1,4507) = 32.2; P = 1.5 × 10⁻⁸]. If only 1st trials were selected (light gray), this correlation was weaker but still significant [light gray broken line, slope = 0.03, adjusted r² = 0.001, F(1,2242) = 4.06; P = 0.04]. This correlation was also significant if only non-1st was included [dark gray, slope = 0.06, adjusted r² = 0.003, F(1,2263) = 7.4; P = 0.006]. At bar release (right), no significant correlation was found between lipping and LC activity (P > 0.05).