Local AMPA receptor blockade in medial shell elicits eating and defensive treading behavior in a rostrocaudal gradient
Localized glutamate disruptions in medial shell induced by microinjections of DNQX, an AMPA/kainate receptor glutamate antagonist, stimulated intense appetitive and/or fearful behaviors depending on placement along a rostrocaudal gradient as expected (). At rostral sites in medial shell, NAc glutamate disruptions generated robust elevations nearly 5-times over vehicle levels in amounts of eating behavior and food consumed during the 1-hr test (cumulative duration of eating: drug × site interaction, F(1,32) = 10.0, p = .003; food intake measured in grams consumed: drug × site interaction, F(1,32) = 14.5, p = .001, , ). Conversely, at caudal sites in medial shell, DNQX microinjections did not elevate food intake (and in some caudal rats actually suppressed eating and food intake below control vehicle levels; ), but instead generated profound elevations in the incidence of fearful distress vocalizations (, ; 73% of rats after DNQX microinjection vs 0% after vehicle, McNemar’s test, p = .001) and of fearful escape attempts to human touch (, ; 40% of rats after DNQX vs 0% after vehicle, McNemar’s test, p = .031). Likewise, caudal DNQX microinjections generated nearly 10-fold increases in the spontaneous emission of defensive treading-burying behavior over vehicle control levels (, ; drug × site interaction in cumulative duration of treading, F(1,32) = 6.9, p = .013, ). Defensive treading typically was not diffuse or random, but rather was directionally focused on a particular target: usually towards the transparent front of the cage (beyond which objects and people in the room could be seen) and towards light-reflecting front corners of the transparent plastic chamber.
Summary maps of behavior and Fos plume analysis
Motivated behavior summary graphs
Effects of D1 and D2 antagonism on DNQX-induced eating and defensive fearful behaviors
D1 dopamine receptor transmission alone needed for DNQX to generate appetitive behaviors at rostral sites
A novel finding here was that endogenous local dopamine stimulation was needed only at D1-like (D1, D5) receptors around the microinjection site in rostral shell for the generation of intense appetitive behavior by DNQX microinjections. Rostral D2-like receptors (D2, D3, D4) appeared essentially irrelevant to glutamate-related amplification of eating behavior and food intake (–). That is, when the dopamine D1-antagonist, SCH23390, was added to the rostral DNQX microinjection, the D1 blockade abolished the ability of DNQX to increase time spent eating or food intake, leaving eating behavior and intake at control levels seen after vehicle microinjections ( and , eating: SCH23390, F(1,7) = 13.3, p = .008; , grams intake: SCH23390, F(1,7) = 11.1, p = .010).
By contrast, combining the D2-like antagonist raclopride with DNQX microinjection for rostral sites failed to prevent or even impair the DNQX-enhancement of eating (cumulative duration; and , raclopride, F(1,8) < 1, p = .743) or food intake (grams consumed; , raclopride, F(1,8) < 1, p = .517). Quite the opposite, at least at caudal shell sites, adding the D2 antagonist allowed caudal DNQX to further increase time spent eating to even higher levels that were 245% above vehicle, or 156% above eating levels produced by DNQX alone (, ; DNQX stimulation of eating at caudal sites was usually low due to the rostrocaudal gradient: average of 566 sec +/− 101 sec on DNQX plus raclopride versus 362 sec on DNQX alone and 230 sec on vehicle; raclopride × DNQX, F(1,10) = 6.0, p = 0.035). A slight caveat to this additional enhancement is that adding the D2 antagonist did not actually boost the physical amount of food consumed for this group, even though it nearly doubled the proportion of time during the trial in which rats ate (, raclopride, F(1,11) < 1, p = .930; however, we note that raclopride did boost stimulation of food consumption as well as of eating behavior for caudal DNQX microinjections in a separate experiment tested below (in tests conducted in a more stressful environment).
As expected, combining both the D1 antagonist and the D2 antagonist together with DNQX completely prevented DNQX from enhancing eating (similar to D1 antagonist above), and kept levels of intake equivalent to vehicle baseline levels (; versus vehicle: grams intake, F(1,7) < 1, p = .973; eating, F(1,7) = 1.1, p = .322). However, the D1–D2 mixture of antagonists was no more effective than adding just the D1 antagonist alone to DNQX, which also completely prevented appetitive increases (; eating, SCH23390 plus raclopride versus SCH23390 alone, F<1, p = 1.000). In short, we conclude that only local endogenous D1 receptor neurotransmission is needed to enable glutamate disruptions in rostral sites of medial shell to stimulate appetitive behavior and food intake. By contrast, local D2 receptor neurotransmission is essentially irrelevant to rostral eating stimulation, being neither necessary nor even contributing additively in any detectable way (and possibly even inhibiting the stimulation of eating at caudal sites, perhaps via generation of fearful reactions as described below that could compete with or suppress appetitive eating).
Ruling out general suppression of appetitive/fearful behavior by dopamine antagonists
Finally, the prevention of DNQX-induced increases in food intake or eating by D1 receptor blockade appeared to reflect a specific interaction of dopamine receptors with glutamate disruptions rather than a general independent suppression of eating motivation or capacity induced by dopamine blockade. Neither microinjections of the D1 antagonist by itself (without DNQX) nor of the D2 antagonist by itself (without DNQX) suppressed baseline levels of eating below control vehicle levels of about 1 gram of chow per session (eating: SCH23390, F(1,14) = 1.9, p = .194, 149 sec +/− 52 SEM on SCH23390 versus 166 sec +/− 54 SEM on vehicle; raclopride: F(1,14) < 1, p = .389, 227 sec +/− 56 SEM; grams intake: SCH23390, F(1,14) < 1, p = .514, 1.15 grams +/− .36 SEM on SCH23390 versus .94 grams +/− .23 SEM on vehicle; raclopride, F(1,14) = 3.9, p = .068, 1.82 grams +/− .42 SEM). Thus local dopamine blockade in NAc at these doses did not impair either normal levels of motivation to eat or the motor capacity for ingestive movements. Instead our results seem to reflect a specific role of D1 receptor dopamine signals in enabling local AMPA receptor glutamate disruptions in rostral shell to stimulate eating behavior to high levels.
Fearful behaviors elicited by local glutamate disruption depend on concurrent local D1 and D2 receptor stimulation from endogenous dopamine
By contrast, simultaneous endogenous signaling at both D1 and D2 receptors in caudal sites of medial shell appeared necessary for DNQX microinjection to generate intense fearful behaviors (–). Mixing either the D1 antagonist or the D2 antagonist with DNQX effectively prevented the production of any defensive treading at caudal sites, as well as the generation of any distress calls or escape reactions to human touch that otherwise were potentiated by DNQX microinjections (, ; defensive treading: SCH23390, F(1,10) = 7.1, p = 0.024, raclopride, F(1,10) = 5.4, p = 0.043; escape attempts & jumps: DNQX alone: 40% of rats, DNQX plus SCH23390: 0%, p = 0.031 [compared to DNQX, McNemar’s test], DNQX plus raclopride: 13%, p = .219; distress calls: DNQX alone: 73% of rats, DNQX plus SCH23390: 13% of rats, p = .012, DNQX plus raclopride: 20% of rats, p = .008). In short, all fearful behaviors remained at near-zero control levels when either dopamine antagonist was mixed with DNQX.
Ruling out general suppression by dopamine antagonist microinjections
Again, D1 and D2 receptor contributions to DNQX fear induction appeared to reflect a specific interaction of these dopamine receptors with the glutamate disruption in caudal shell, because giving microinjections of either or both dopamine antagonists in the absence of DNQX did not change defensive treading from vehicle baseline levels (treading: SCH23390, F(1,14) < 1, p = .913; raclopride, F(1,14) < 1, p = .476). However, it must be noted that vehicle levels of fearful behaviors were near zero already, raising the possibility that a floor effect could have obscured a general suppression of fearful behavior by dopamine blockade. Therefore we turn to other evidence, which also suggests that dopamine antagonist microinjections, either with DNQX or by themselves, did not generally prevent most behaviors. For example, grooming, a nonvalenced behavior that was emitted at substantial rates after vehicle, remained unsuppressed by local blockade of D1 or D2 receptors. Dopamine antagonists alone did not suppress spontaneous grooming (average of 9.33 +/− 1.35 bouts on vehicle versus 8.09 +/− 1.13 on SCH23390 and 8.40 +/− 1.22 on raclopride; F<1). Likewise, adding dopamine antagonists to DNQX did not suppress grooming behavior (F<1). Microinjections of the dopamine antagonists alone did moderately suppress locomotion expressed as rears and cage crosses by about 50% from vehicle levels, though this suppression was nowhere near as strong as the abolition of DNQX-induced elevations of eating or fearful defensive treading described above (rears: SCH23390, F(1,13) = 17.6, p = .001, raclopride, F(1,13) = 9.8, p = .008; cage crosses: SCH23390, F(1,13) = 19.3, p < .001, raclopride, F(1,13) = 13.1, p = .002). Further, DNQX microinjections stimulated locomotion to double or triple vehicle levels, and adding SCH23390 or raclopride to the DNQX microinjection did not prevent that rise in cage crosses and rears (main effect of DNQX: cage crosses, F(1,33) = 12.0, p = .002; rears, F(1,33) = 6.8, p = .014; SCH23390: F<1 for rears and cage crosses; raclopride: cage crosses, F(1,19) = 2.2, p = .154; rears, F(1,19) = 3.2, p = .091). Thus general suppression effects of dopamine antagonists were either missing or minimal, and did not appear sufficient to account for the abolition of DNQX-stimulated motivated behaviors described above.
Local mode of dopamine-glutamate interaction switches flexibly as ambience reverses motivation valence
Environmental ambience flips motivational valence
As expected, for most sites in the intermediate two-thirds of medial shell (i.e., all sites between far rostral 20% and far caudal 20%), changing environmental ambience from dark, quiet and familiar (similar to rats’ home-room) to stressfully bright and noisy (extra light and raucous music) reversed the valence of motivated behavior generated by DNQX microinjections (Reynolds and Berridge, 2008
) (). Rats emitted almost exclusively appetitive behavior in the Home environment after DNQX microinjections, but emitted substantial amounts of fearful behaviors as well when tested in the Stressful environment after DNQX at the same NAc sites. The familiar, low-stimulation and presumably comfortable conditions of the Home environment (which rats have been shown to prefer to standard lab illumination condition; Reynolds and Berridge, 2008
) caused the appetitive-stimulating zone within NAc to expand from rostral sites and invade caudal sites of medial shell as well, so that 90% of all medial shell locations generated intense eating behavior and food intake (greater than 200% of vehicle; ). Concomitantly, the Home environment virtually eliminated DNQX-induction of fearful behaviors, such as distress vocalizations, escape attempts or defensive treading (; treading, DNQX, F(1,7) = 3.5, p = .102; drug × site interaction, F(1,7) < 1, p = .476). Consequently, the size of the fear-inducing zone severely shrank in the Home environment, leaving most mid-caudal sites unable to generate fearful reactions. Thus only one rat (which had the farthest caudal shell site) displayed more than 20 seconds of defensive treading in the Home environment, or emitted a distress vocalization when touched after the test ().
Environmental ambience shifts glutamate-dopamine interaction mode
In contrast, the loud and bright Stressful environment (which rats avoid over lab conditions and quickly learn to turn off when given the opportunity; Reynolds and Berridge, 2008
) expanded the caudal fear-inducing zone to include substantial mid-rostral areas of medial shell, and increased the levels of defensive treading stimulated by DNQX to over 600% the corresponding levels induced in the Home environment (; DNQX, F(1,7) = 23.8, p = .002; site × drug interaction, F(1,7) < 1, p = .429). Similarly, the Stressful environment increased the incidence of distress vocalizations generated after DNQX when the rats were touched by the experimenter at the end of the session by five-fold compared to the Home environment (; 50% of rats versus 10% at Home; McNemar’s test, p = .063). Conversely, the Stressful environment eliminated pure appetitive sites in the mid rostrocaudal zone, converting them into either mixed valence or purely fearful sites (). The Stressful environment also reduced the intensity of appetitive behaviors induced by DNQX at midrostral sites to approximately 50% of Home levels, even for sites that still generated any eating (average of 507 sec +/− 142 SEM in the Stressful Environment versus 879 sec +/− 87 SEM in the Home Environment; drug × environment interaction, eating, F(1,7) = 6.0, p = .044; food intake, F(1,7) = 2.9, p = .013).
Fearful mode requires D2 receptor involvement, but appetitive mode does not
The most important novel finding here was that D1/D2 receptor requirements for endogenous dopamine stimulation at a given site dynamically changed with environmental ambience shifts in a manner tied to motivational valence generated by DNQX at the moment rather than to rostrocaudal location per se. Each DNQX site had two modes: appetitive and fearful, depending on external ambience of the moment. The appetitive mode (i.e. DNQX-stimulation of eating induced by the dark, quiet and familiar Home environment) did not require D2 receptor activation to enhance eating, whereas the fearful mode (i.e. DNQX-stimulation of defensive treading behavior and distress vocalizations induced by the loud and bright Stressful environment) always required D2 receptor activation for every site to stimulate fear, regardless of rostrocaudal location (just as caudal sites had required D2 for DNQX generation of fear in the previous experiment) (). Flips in valence mode, between appetitive and defensive, occurred for 90% of sites tested, which comprised nearly all possible intermediate rostrocaudal locations in medial shell. For the remaining 10% of sites (n = 1), DNQX microinjected into far caudal shell always generated fearful behaviors in both environments (and fearful behaviors were always eliminated by D2 blockade).
More specifically, adding the D2 antagonist to DNQX microinjection completely blocked distress calls and defensive treading behavior at all sites that otherwise generated fear after DNQX in the Stressful environment (; rostral sites, raclopride, F(1,4) = 19.9, p = .021, all rats, raclopride, F(1,7) = 10.7, p = .022, site × drug interaction, F(1,7) < 1, p = .730). However, the D2 antagonist never blocked or suppressed eating behavior (i.e., appetitive motivation) generated at the same sites by DNQX in the Home environment; in fact, adding the D2 antagonist actually enhanced the levels of eating behavior generated by DNQX in the Stressful environment to 463% of vehicle levels and 140% of levels on DNQX alone for the same sites (; average of 712 sec +/− 178 SEM on DNQX plus raclopride versus 507 sec on DNQX alone and 153 sec on vehicle). In the Stressful environment, D2 blockade magnified DNQX-stimulation of eating and increased grams of food consumed, regardless of rostrocaudal location (within the intermediate zone), confirming that local D2 neurotransmission is not only unnecessary for eating enhancement but actually can oppose the generation of intense eating by local AMPA receptor blockade in medial shell (eating, raclopride, F(1,7) = 18.5, p = .008; site × drug interaction, F(1,7) < 1, p = .651; food intake, raclopride, F(1,7) = 5.6, p = .064, site × drug interaction, F(1,6) = 2.5, p = .163). While in the Standard environment D2 blockade disinhibited DNQX-eating only in caudal shell (), the Stressful environment expanded the fear generating zone and likewise expanded the zone in which D2-blockade disinhibits DNQX-eating to include mid-rostral zones of medial shell (; eating, raclopride × environment × site interaction, F(1,25) = 6.2, p = .020).
Dopamine receptor roles flip reversibly between multiple transitions
In rats that displayed ambivalent (both) motivations in the Stressful environment (60% of rats), DNQX-induced eating peaked in the first 15 minutes, while defensive treading peaked later in the trial (30 – 45 minutes after the microinjection, ). During the 20 minutes period of maximal overlap between appetitive and defensive behavior (minutes 10 – 30), most rats transitioned from appetitive to defensive only once (16%) or 2 to 6 times (50%). With relatively few transitions during the hour, any single minute was likely to consist of pure rather than mixed motivated behaviors (), consistent with previous reports (Reynolds and Berridge, 2008
). Dopamine D2 receptor blockade did not block eating behavior (which dominated in the first 20 minutes of the session), but effectively blocked defensive treading behavior (which dominated in the final 20 minutes).
Appetitive and defensive behavior elicited from mixed valence sites in the Stressful environment
However, two rats stood out as especially ambivalent, transitioning between appetitive and defensive behavior more than 25 times each within the hour after pure DNQX microinjections in the Stressful environment. This represented the closest approach to simultaneous display of opposite motivations that we observed. Even in these rats, however, D2 receptor blockade consistently blocked only defensive behavior emitted under the loud and bright conditions, and never appetitive behavior (in either Stressful or Home environments) (example rat, ) which continued to occur at similar levels and time points after DNQX plus D2 antagonist microinjection as after pure DNQX in the corresponding environment. Thus motivated behavior produced by dopamine-glutamate interactions appeared to be able shift rapidly and repeatedly between appetitive and fearful modes. When environmental conditions fostered ambivalence in a susceptible individual, a site could flip valence modes more than 20 times in a single hour.
Fos plume analysis: defining size of microinjection local impact
Localization of function was aided by assessing the extent of local impact of drug microinjections on nearby tissue, as reflected in Fos plumes around the microinjection center (). Rats used previously for behavioral testing in the environmental shift group were assessed for Fos plumes after the end of the experiment. However, as anticipated, we confirmed that rats that had already completed behavioral testing had shrunken Fos plumes compared to the dedicated Fos group that received only a single microinjection, indicating that DNQX-induced plumes from rats that received 6 previous microinjections no longer represent the maximal impact radius of drug spread. DNQX produced plumes in the dedicated Fos group that were nearly 4 times larger in volume (nearly 2 times larger in radius) than in the previously behaviorally-tested group (F(9,90) = 3.3, p < .002). Therefore, when mapping functional drug spread in all figures, we relied on plume radius data from the dedicated Fos group (matched to initial behavioral test conditions) to avoid underestimation when assessing the maximal spread of local impact for microinjections, and to construct plume maps for localization of function. However, all other data besides plume radii shown in maps were obtained exclusively from the behaviorally-tested group (i.e., colors and bar graphs reflecting intensities of eating and fearful behaviors induced at particular sites).
Pure DNQX microinjections produced plume centers of double the intensity of vehicle-level Fos expression, in a small volume of 0.02 mm3
for the dedicated Fos group (, top middle; radius = 0.18 +/− 0.04 mm SEM). Rats that had received 6 previous microinjections had an even smaller volume center of 0.004 mm3
(radius = 0.1 mm). Surrounding plume centers, Fos expression in the maximal group had a larger halo of 0.23 mm3
volume of milder elevation >1.5 times vehicle levels (radius = 0.38 +/− 0.05 mm SEM; rats previously tested 6 times had smaller outer halos of 0.05 mm3
volume, radius = .23 mm). Addition of the D1 antagonist (SCH23390) shrank plumes and attenuated
the intensity of DNQX-induced elevations in local Fos expression (, bottom middle; DNQX versus DNQX plus SCH23390, Post hoc pairwise comparison with Sidak corrections, p < 0.01). SCH23390 shrank the total volume of DNQX Fos plumes to less than 0.18mm3
(outer halo radius = 0.35 +/− 0.05 mm SEM). By contrast, addition of the D2 antagonist (raclopride) expanded intense centers of Fos expression and enhanced
DNQX-induced elevation in local Fos expression (, bottom left; DNQX versus DNQX plus raclopride, Post hoc pairwise comparisons with Sidak corrections, p < 0.05). Raclopride expanded the inner center of doubled Fos expression produced by DNQX to a volume of 0.15 mm3
(radius = .33 +/− 0.042 mm SEM), and left unchanged the radius and intensity of the outer plume halo (of 1.5x expression). We note that the D1 antagonist apparently predominates over the D2 antagonist in effects on local Fos when both are microinjected jointly with DNQX, as DNQX Fos plumes shrink following the addition of combined D1 and D2 antagonists (Faure et al., 2008