CRF and amphetamine in nucleus accumbens increase cue-triggered lever pressing associated with sucrose reward
CRF (high dose 500 ng/0.2 μl; low dose 250 ng/0.2 μl) and amphetamine (20 μg/0.2 μl) microinjections were located in the region of medial shell of nucleus accumbens that contains highest CRF levels (Figure ; Figure ). CRF and amphetamine each dramatically and similarly increased cue triggered incentive motivation to obtain reward (Three-way ANOVA (drug × cue presence × lever), F3,159 = 5.57; p < 0.05, main effect of drug). Both CRF and amphetamine comparably increased phasic CS+-triggered peaks of increased pressing on the lever that previously had been associated with sucrose reward compared to vehicle, but did not increase lever pressing at other times (i.e., during CS- or in the absence of any CS) (Two-way ANOVA (drug × cue presence), F3,79 = 4.21; p < 0.05, significant interaction) (Figure ).
Figure 1 Fos plumes. Coronal sections showing point sample positions used to identify local Fos plumes around microinjection site. A. Vehicle microinjection produces merely low elevation of Fos compared to normal virgin tissue. Radial arms extending from center (more ...)
Figure 2 Fos plume maps of CRF (500 ng) amplification of cue-triggered increases in lever pressing. Fos plume maps of functional localization: CRF effects on cue-triggered incentive salience. Sagittal (A), horizontal (B), and coronal maps (C) show each Fos plume (more ...)
Figure 3 Enhancement of incentive motivation by CRF. Effects on cue-triggered lever pressing during extinction test caused by CRF (250 and 500 ng) and amphetamine (20 μg) microinjections in the caudal medial accumbens shell. A. Lines show pressing in the (more ...)
Post-hoc comparisons indicated that CRF enhancement of cue-triggered peaks of lever pressing was exclusively due to the highest dose of CRF (500 ng; Bonferroni, p < 0.05). Specifically, 500 ng CRF microinjections tripled the amplitude of phasic peaks in pressing on the sucrose-associated lever during the 30-sec CS+ tone or clicker and in the 30-sec period immediately after it, compared to the same lever after vehicle microinjections (Figure ), but not during other periods. An equivalent tripling of cue-triggered peaks in lever pressing was also produced by amphetamine (20 μg) microinjections at the same sites (Bonferroni, p < 0.05) (Figure ), used as a standard here for enhanced cue-triggered 'wanting', and confirming previous results [30
]. By contrast, the low dose of CRF (250 ng) failed to significantly enhance active lever pressing over vehicle despite the possibility of a slight trend (Bonferroni, p = 0.42, n.s), and by itself the 30-sec CS+ elicited merely a modest rise of only 50% over pre-cue baseline in lever pressing after vehicle microinjections, which was less than half the maximum drug-enhanced levels.
Associative cue specificity: CS+ vs baseline
In order to rule out motor arousal and general frustration effects of CRF as explanations of increased peaks in incentive motivation, it is crucial that enhancement not also apply to the plateaus of lever-pressing during periods between cue-triggered peaks. Motor arousal and aversive effects can be caused by intracerebral microinjections CRF [38
]. However, motor arousal and frustration effects of CRF would be expected to remain relatively constant over a 30 min test, or at least last longer than the minute or so impact of a cue. Thus if motor arousal or aversive states were the explanation for elevated instrumental behavior here, lever pressing during baseline periods in between auditory stimuli should have been elevated too – not only when a CS+ was present. However, neither CRF (500 or 250 ng) nor amphetamine (20 μg) produced detectable increases in baseline instrumental responding during no-stimulus baseline periods (One-way ANOVA (drug), F3,39
= 0.3, p = 0.8, n.s.). Baseline instrumental responding in the absence of any CS (i.e., pre-cue periods) was never increased by any microinjection (even when it increased lever pressing in response to the CS+). This dissociation between enhanced cue-triggered incentive motivation during CS+ versus unchanged baseline responding was separately confirmed in a two-way ANOVA by a significant interaction between the presence/absence of CS+ and administration of either CRF (drug × cue presence interaction, F2,119
= 5.23, p < 0.05) or amphetamine (F1,79
= 4.25, p < 0.05) (Figure ).
Associative cue specificity: CS+ vs. CS-
Similarly, the CS+ (tone or click) was the only conditioned stimulus that ever significantly increased lever pressing on the sucrose associated lever (Three-way ANOVA (cue type × drug × lever), F1,119 = 10.40, p < 0.001, main effect of cue type; Figure ). The control CS- (a different auditory stimulus that did not predict sucrose reward) failed to increase lever pressing over the baseline pre-cue period in any drug condition (Figure ). That selective pattern of only-CS+ enhancement is required by the hypothesis of incentive salience magnification, which posits that enhanced incentive salience is attributed specifically to stimuli previously associated with reward.
A useful way of further assessing the associative specificity of CS+ effects versus CS- effects on Pavlovian-Instrumental transfer effects is to calculate transfer scores between them. A transfer score directly contrasts pressing during CS+ versus CS- by subtracting CS- pressing from CS+ pressing on a within-subject basis, so that the remainder shows the enhanced CS+ effect for each individual rat. When we calculated this, the resulting transfer scores confirmed that both the CRF high dose (500 ng) and amphetamine (20 μg) microinjections significantly and specifically amplified the incentive effect of the CS+ on pressing the sucrose-associated lever, compared to vehicle and low dose CRF microinjections (Figure ).
Thus, CRF (500 ng) and amphetamine (20 μg) magnification of cue-triggered peaks in lever pressing applied only to the CS+ that previously was associated with sucrose UCS, and not to the control CS- which did not signal sucrose.
Temporal reversibility of cue-triggered pursuit: dependence on CS+ presence
If increased lever pressing for reward is caused by excessive attribution of incentive salience to the reward's CS+, then the temporal pattern of peaks of cue-triggered responding should be phasic, reversible, and repeatable [30
]. In other words, peak bursts of active lever pressing should come and go with successive presentations of the 30-sec CS+, and disappear again soon after each presentation. A temporal analysis of responding confirmed this pattern for CRF (500 ng) and amphetamine (20 μg) enhancement of pressing on the sucrose-associated lever during CS+ presentations (Figure ). A magnified peak of pressing on the lever previously associated with sucrose reward was triggered anew by each presentation of the CS+ sucrose cue after CRF (500) or amphetamine microinjections, maximally elevating during its 30-sec period (Two-way ANOVA (cue presence × cue presentation order), F1,79
= 16.11, p < 0.001; main effect of cue presence). Pressing rose within seconds of each CS+ and remained dramatically elevated throughout the 30 sec presentation. After almost every CS+, pressing decayed significantly within 1 min of its end, and always descended to baseline levels again before the next CS occurred 4 min later.
Figure 4 Temporal pattern of lever pressing within test session. Minute-by-minute time-course of lever pressing peaks on sucrose-associated lever after microinjections of (A) vehicle, (B) amphetamine 20 μg, (C) CRF 500 ng, and (D) CRF 250 ng. Open circles (more ...)
This temporal pattern of cue-triggered phasic peaks of pressing (without enhancing intervening plateaus or CS- pressing) is important because it further rules out alternative explanations mentioned above, based on traditional CRF/amphetamine effects that have more constant time course durations (i.e., on the order of several minutes or more). Those include increased motor arousal, general impulsiveness or aversive states that would last longer than 30 sec, and so should be present during no-stimulus baseline periods (and CS- presentations) between CS+ tones. By contrast, the enhanced incentive salience interpretation specifies that CRF should specifically amplify attribution only to the reward CS+, just as amphetamine microinjection does, and so seems to fit best this observed pattern of effects.
Target specificity of cue-triggered incentive motivation: active versus inactive levers
Cue-triggered increases in efforts to obtain sucrose were directed specifically to the active lever that had previously earned sucrose reward during training, and not to the other lever (Two-way ANOVA (drug × lever), F3,79 = 3.25; p < 0.05, significant interaction; Figure ). Rats made few responses on the second control lever that had never been associated with sucrose reward. Pressing on the control lever was not increased during the CS+ by either CRF (500 ng) (Two-way ANOVA (drug × cue presence), F2,59 = 0.35, p = 0.55, n.s.) or amphetamine (20 μg) (Two-way ANOVA (drug × cue presence), F2,59 = 0.36, p = 0.55, n.s.). Lever specificity was further confirmed by finding a significant difference between previously-active lever versus always-inactive lever in pressing during CS+ (Two-way ANOVA, F1,159 = 5.12, p < 0.05), and a significant interaction between lever identity and drug in the microinjection-induced enhancement of cue triggered increases in pressing (Two-way ANOVA (drug × lever), F1,159 = 34.15; p < 0.001). Thus, the CS+ sucrose cue selectively triggered increased pressing only on the lever previously associated with obtaining sucrose. That suggests that our CRF incentive on PIT lever pressing are not explained by nonspecific motor arousal induced by CRF, or a general sensorimotor tendency to emit more pressing movements regardless of target, even if CRF produced any motor or arousal effects (Figure ).
Pavlovian approach CRs to sucrose dish
Presentations of CS+ also elicited Pavlovian approach conditioned responses to the sucrose dish (especially after vehicle microinjections), either as a Pavlovian S-R habit elicited by the CS+, or as a discriminative instrumental response to earn sucrose. However, CRF and amphetamine microinjections both suppressed conditioned dish approach below the vehicle control level (F3,159
= 2.79, p < 0.05; Figure ). That suppression is noteworthy because it helps rule out the several remaining alternative explanations. For example, approach suppression by CRF indicates that the enhancement of PIT cannot be due to CRF potentiation of S-R Pavlovian habits, because dish approach was the only S-R habit ever actually paired with CS+ tone during training. Similarly, approach suppression indicates that CRF did not potentiate discriminative instrumental responding (in which the CS+ might have acted as a discriminative stimulus (SD
) to signal that instrumental approach would be reinforced, and this instrumental relation might later have been generalized to lever pressing [41
] because any such instrumental relation between approach and reinforcement was actually suppressed by CRF and amphetamine during the PIT test, and not enhanced. Sucrose dish entries during the first CS+ were especially suppressed by both CRF doses (Two-way ANOVA (drug × cue presence), F2,79
= 4.21, p < 0.01). Sucrose dish approaches were similarly suppressed by amphetamine microinjection (F1,39
= 6.40, p < 0.05). Suppression of dish approach during the CS+ by CRF and amphetamine may have occurred possibly as a secondary consequence of increased response competition from higher cue-triggered rates of lever pressing that occurred at the same time.
Figure 5 Response competition between sucrose dish approach and lever pressing. A. Averaged time-course of approach responses to sucrose dish just before, during and after first CS+ cue presentation. B. Same information for pressing on sucrose-associated lever. (more ...)
The suppressive effects on dish approach CRs after microinjection of CRF or amphetamine were associatively specific to the CS+ (drug × CS+ interaction, F1,39 = 6.40, p < 0.05) (Fig. ), and the same was true for amphetamine microinjection (drug × CS+ interaction, F1,39 = 5.34, p < 0.05). Neither CRF nor amphetamine microinjections decreased food cup entries below vehicle levels during the baseline 30 s pre-cue periods (2-way ANOVA, significant interaction (drug × cue presence), F3,79 = 3.47, p < 0.05) or during CS- presentations (drug × CS+ interaction, F1,39 = 3,25, p = 0.68, n.s.).
Finally, we note that CRF effects on PIT versus dish approach were independent and partially dissociable by dose: both 250 ng and 500 ng CRF doses suppressed cue-triggered dish approach during CS+ presentations early in a session, but only 500 ng CRF increased lever pressing. That indicates that suppression of dish approach is not a sufficient cause of CRF enhancement for cue-triggered lever pressing, which appears to require independent incentive motivation mechanisms recruited by the higher dose.
Fos plumes: identifying zones of local neuronal activation
To help identify where our CRF microinjections actually acted in the brain we used a Fos plume mapping procedure to visualize spread of neuronal gene transcription triggered by 500 ng CRF in tissue around microinjection sites [43
]. The local expression of Fos protein is a useful marker for geographic spread of pharmacological impact, even if c-fos transcription is not invariably tied to neuronal activation, at least for drugs that induce acute Fos transcription immediately around a microinjection site (including CRF). Measuring the size of the resulting local Fos plume caused by a microinjection objectively assesses the spherical volume of tissue and intensity of neuronal response impacted by local drug action. Fos expression in a local neuron might either reflect direct drug action on receptors on that neuron, or else reflect indirect action from adjacent neurons containing receptors that act on Fos-expression neurons via local circuits. In either case, the Fos plume reflects a local sphere of functional modulation induced by CRF microinjection, and provides quantitative information on its intensity and size. The boundaries of the plume reveal where the drug stops having an intense functional impact, even if drug molecules spread further beyond the plume in lower concentrations insufficient to trigger gene transcription.
For microinjections of CRF (500 ng) we identified two zones of elevated Fos expression: intense versus moderate elevation zones (Figure ). Each zone was defined by 2 independent criteria. An inner zone of intense Fos elevation was defined as the mean radius within which 1) absolute Fos expression was increased by at least 1 order of magnitude over normal tissue levels (10 times spontaneous levels in medial shell), and 2) relative Fos expression was increased by at least twice over the levels produced at equivalent locations after vehicle microinjections (2 times vehicle-induced levels). An outer zone of moderate Fos elevation was defined at the mean radius within which 1) absolute Fos expression was increased by 3 times (but not 10 times) over normal tissue levels, and 2) relative Fos expression was increased by 5 times over the level of equivalent points after vehicle locations (note: the reason why our outer zone used a higher vehicle-relative threshold than the inner zone was that a vehicle microinjection induces some Fos expression in a small centrally-restricted inner zone, perhaps due to pressure of the microinjection or irritation from cannula-induced damage. The resulting slight elevation in inner zone vehicle baseline [above normal tissue baseline] imposed a ceiling on drug-induced Fos expression in this central zone that limited relative increases to under 5× [even if drug caused a >10× absolute increase]).
The mean inner zone of intense Fos elevation produced by CRF microinjection at the behaviorally most effective dose (500 ng) was approximately 0.25 mm in radius (absolute 10× increase over normal; relative 2× increase over vehicle) and the outer zone of low elevation was an additional 0.2 mm radius (absolute 5× increase over normal; relative 5× increase over vehicle; Figure ). Therefore, to represent these zones we assigned color-coded symbols of corresponding 0.25 mm radius in size to represent each inner zone, surrounded by similarly colored but more pale halos of additional 0.2 mm radius to represent each outer zone. Thus symbols and halos served to bracket the estimated range of intense-moderate functional activation likely to mediate the mapped microinjection effects on behavior.
To calculate the 3-dimensional plume volume produced by CRF 500 ng microinjections, we assumed a roughly spherical shape and calculated the inner intense sphere to contain a volume of approximately 0.06 mm3, and the outer sphere to contain a volume of approximately 0.38 mm3. Because the entire medial shell is approximately 2.87 mm3 in total volume, these volumes meant that the inner sphere of a CRF (500 ng) plume filled approximately 2% of total medial shell volume, whereas its outer sphere filled approximately 13%. Of course, some microinjections near borders may not have filled the shell to quite that extent if they partially penetrated other structures such as the medial core (though adjacent penetration was never deeper than 0.5 mm).
We mapped the extent of activation diffusion as reflected by Fos plume around each microinjection site. In order to further assess the role of diffusion into other structures or the ventricles, we took two additional steps. First, we compared PIT effects for 'hits' in nucleus accumbens shell versus 'missed' anatomical control sites in lateral septum dorsal to the nucleus accumbens. No enhancement of PIT by CRF or amphetamine was evident in rats with dorsal anatomical control sites outside the nucleus accumbens (Figure ), indicating that the PIT enhancement effects described above was not due to diffusion upwards along cannulae tracks to dorsal structures. Second, we inspected individual brain slices to identify any sites in medial shell where the Fos plume could touch the wall of the lateral ventricle. This seemed worth doing because intra-ventricular CRF administration in the 1000 ng range has been reported to produce arousal and conditioned aversion effects, and so conceivably might have been responsible also for PIT magnification effects here. To assess if leakage into ventricles was the primary source of CRF effects here, we compared magnitude of PIT enhancement by CRF in rats with Fos plumes that touched the ventricle wall versus the group of rats with plumes contained entirely within the nucleus accumbens. Two rats had plumes that definitely touched lateral ventricle, and another two had plumes that might have touched, whereas 6 rats had plumes fully contained away from the ventricle wall. By themselves, the group of 4 rats with ventricle-touching plumes did not show a significant PIT enhancement effect by CRF (mean score 2.7, SEM = 1.3; F1,7 = 2.03, p = 0.20). By contrast, the group with non-touching plumes contained fully within nucleus accumbens away did show an enhancement of PIT by CRF microinjection (mean score 3.33; SEM = 1.17; F1,63 = 10,17, p < 0.05). That pattern across groups indicates that the significant PIT enhancement effects described above were not driven primarily by CRF ventricle diffusion in this group. Finally, we found no significant difference between these two groups (F1,9 = 0.10, p = 0.75), indicating that most sites tested in nucleus accumbens contributed comparably to the PIT effects described above.
Functional Fos plume mapping of microinjection effects on behavior
Functional site effects were mapped for all cannulae placements in nucleus accumbens and adjacent structures (Figure ). Colors for symbols and halos represented the magnitude of behavioral effects produced by CRF (500 ng) microinjections at each microinjection site change score in elevation of pressing on sucrose-associated lever minus to the control vehicle effect at that same site in the same rat). We chose the sagittal plane primarily to map Fos plumes and functions because sagittal view allows the entire rostrocaudal and dorsoventral extents of medial shell to be viewed on a single atlas map (Figure ). Additional supplemental maps were also constructed in coronal and horizontal planes to allow full 3-D mapping of functional effects [46
The Fos plume maps for localization of function showed that most nucleus accumbens microinjections were successfully placed in the intended posterior-central zone of medial shell that has highest levels of CRF receptors and terminals, and within that target zone most CRF (500 ng) microinjections produced comparable magnifications of PIT (Figure ). The spread of local Fos plumes did not otherwise extend to other structures around the medial shell, including dorsal structures, except that a few microinjections may have penetrated approximately 0.4 mm into the medial border of nucleus accumbens core.
Although more remains to be done in future regarding localization of CRF function for incentive motivation in nucleus accumbens and related structures, these initial maps indicate that CRF activation in our present study was restricted to the nucleus accumbens medial shell, and possibly a small strip of adjacent core. Within that zone of medial shell, CRF activation appears sufficient to magnify the level of incentive salience attributed to a CS+ that was previously associated with reward (Figure ).