Fos plume analysis of local impact
Fos plumes were used as a tool to map the spread of drug impact on neuronal function immediately around the microinjection site. This mapping tool provides objective information on where a microinjection directly influences gene transcription/translation in local neurons and where its influence ends. DNQX (450 ng) quadrupled local Fos expression over vehicle levels in an ~<0.08 mm3
volume plume center of the microinjection site, which was surrounded by less intense doubling of Fos expression in a larger plume halo of ~3.9 mm3
volume (). Addition of D1
antagonists to the mixture containing DNQX markedly reduced the volume and intensity of Fos plumes. By themselves, microinjections of D1
antagonists induced only small and faint Fos plumes, just slightly above control vehicle microinjection levels (supplemental Fig. 1
, available at www.jneurosci.org
as supplemental material
Figure 1 Colored plume maps show average local elevations of Fos caused by microinjection of DNQX, relative to vehicle (top left). DNQX caused robust plumes of up to a 4 mm 3 volume, elevated 200 to 400% above vehicle levels. The DNQX minus mixture reduction map (more ...)
That is, in a typical Fos plume, pure DNQX intensely stimulated Fos to more than four times above vehicle control levels in an intense inner center of 0.267 mm radius ± 0.042 mm SEM (corresponding to 0.08 mm3
spherical volume; volume, 4/3 π radius3
). The intense center was surrounded by an intermediate halo with a 0.528 mm radius (±0.068) of more than three times Fos elevation over vehicle (0.6 mm3
sphere volume), and by an outer halo with a 0.978 mm radius (±0.122) of more than two times Fos elevation (3.9 mm3
sphere volume). We note that these 450-ng-dose DNQX plumes were larger than those produced by a lower 250 ng DNQX dose in a recent study that used otherwise similar microinjections (Reynolds and Berridge, 2008
), with a center radius here twice that of the previous lower dose (producing a center volume approximately seven times greater), and an outer radius ~30% longer (producing an outer volume roughly twice as great) for a 450 ng dose than for the previous 250 ng dose. By comparison, adding D1
dopamine antagonists to DNQX microinjections shrunk the centers and middles of mixture plumes to under one-half the size of pure-DNQX plumes (center radius, 0.1 mm; volume, 0.004 mm3
; middle radius, 0.26 mm; volume, 0.03 mm3
), and shrunk the outer plume radius by 30% (0.6 mm) and the total outer volume (0.9 mm3
) to under one-fourth the size of pure DNQX levels. Finally, D1
dopamine antagonists by themselves produced small faint plumes that reached only 0.4 mm3
outer volume. Plume symbols on maps were scaled to match the size of DNQX-induced Fos plumes, with corresponding centers of 0.53 mm in diameter and surrounding halos of 1−2 mm in diameter, and color coded to show behavioral effects evoked at each site (, ).
Figure 2 A, B, Fos plume maps of appetitive eating behavior versus fearful defensive treading behavior generated by DNQX and mixture microinjections. Each plume-sized circle represents color-coded behavioral effects of DNQX or mixture at that site, compared with (more ...)
Figure 4 Summary map of “desire versus dread” motivations produced by microinjections of DNQX versus mixture (D1/D2/DNQX). Appetitive eating behavior (green symbols) was stimulated by DNQX microinjections in the rostral shell, whereas fearful defensive (more ...)
AMPA/kainate receptor blockade in the shell elicited rostrocaudal gradient of eating versus defensive treading behaviors
DNQX microinjections elicited robust fearful versus feeding behaviors along a rostrocaudal anatomical gradient in medial shell of the nucleus accumbens, compatible with previous findings: caudal sites generated fearful defensive treading behavior and rostral sites generated appetitive eating behavior (Reynolds and Berridge, 2003
) (site × drug interaction, F(1,21)
= 5.587; p
= 0.028) (-). The addition of a dopamine-blocking mixture of D1
antagonists to the same microinjection completely blocked DNQX-generation of any increases in either appetitive/consummatory behaviors or defensive/fearful behaviors. Thus, no rostrocaudal gradient was detectable after dopamine blockade, and DNQX failed to increase motivated behaviors of any type in the absence of local dopamine neurotrans-mission. Finally, isotonic saline and DMSO/saline vehicles never significantly altered any motivated or locomotor behavior [defensive treading, eating, drinking, intake, rearing, locomotion; all F(1,10)
< 1, not significant (ns)], and so were always similar to each other in lacking behavioral effects ().
Figure 3 A, B, Magnitude of increases in food intake (A) and defensive treading (B) behaviors elicited by DNQX, mixture, dopamine antagonists alone, or vehicle microinjections in the anterior and posterior halves of the medial shell of the nucleus accumbens. Data (more ...)
Rostral DNQX alone: eating induced by AMPA glutamate receptor blockade
Rostral microinjections of DNQX more than tripled food intake (average of 5.2 ± 0.52 g SEM instead of 1.46 ± 0.57 g SEM under vehicle) and more than doubled the duration of time spent in eating behavior (average of 795 ± 123 s SEM instead of 295 ± 117 s SEM under vehicle; duration, F(1,21) = 6.555, p = 0.018; intake in grams, F(1,21) = 12.487, p = 0.002; sites rostral to +1.8 mm bregma). Rostral stimulation of eating appeared especially potent at dorsal sites in the rostral half of medial shell, compared with more ventral sites (). In contrast, DNQX microinjections at sites caudal to +1.0 mm bregma either decreased eating or failed to change eating behavior (rostral vs caudal, duration, F(1,22) = 11.697, p = 0.003; intake in grams, F(1,22) = 7.383, p = 0.013).
Dopamine antagonists prevent rostral elicitation of appetitive eating
Adding dopamine antagonists to the microinjection containing DNQX (referred to throughout as the mixture condition) completely blocked the increase in eating behavior normally produced by DNQX by itself (DNQX with dopamine antagonists vs DNQX alone: eating duration, F(1,21) = 7.231, p = 0.014; food intake, F(1,21) = 11.163, p = 0.003) (-). Microinjections of the DA-AMPA antagonist mixture in the nucleus accumbens shell produced no change in food intake compared with vehicle condition (eating duration, F(1,21) = 0.240, ns; food intake, F(1,21) = 0.904, n.s). The mixture failed to change eating duration or intake amount even at rostral sites, compared with vehicle-control levels (eating duration, F(1,8) = 1.713, p = 0.227; food intake, F(1,8) = 1.571, p = 0.245), and there was no drug by site interaction (eating in seconds, F(1,21) = 2.603, ns; eating in grams, F(1,21) = 1.189, n.s).
Results from the additional control group indicated that microinjection of the D1
dopamine antagonists alone did not suppress spontaneous eating behavior below vehicle levels in the absence of DNQX at either caudal sites (eating duration, F(1,4)
= 2.054, ns; food intake, F(1,4)
= 1.643, ns) or rostral sites (eating duration, F(1,6)
< 1, ns; food intake, F(1,6)
= 1.064, ns) (). Failure of dopamine blockade in shell by itself to prevent eating is consistent with previous reports that microinjection of SCH-23390 or raclopride in the medial accumbens does not suppress food intake (Baldo et al., 2002
) or powerfully suppress global activity (Yun et al., 2004
). It suggests that dopamine antagonist microinjections did not simply reduce the ability to perform eating movements or to consume food. Rather than simply making rats unable to exhibit motivated behaviors because of motor in-capacity or sedation, local dopamine blockade instead appeared to prevent DNQX from generating intense appetitive eating or defensive treading behaviors, in accordance with the dopamine–glutamate modulation hypothesis.
Caudal DNQX alone: defensive treading induced by AMPA glutamate receptor blockade
DNQX microinjections produced more than a 10-fold increase over vehicle control levels in fearful patterns of defensive treading at medial shell sites caudal to +1.4 mm bregma, following a rostrocaudal gradient for defensive behavior in the nucleus accumbens shell (Reynolds and Berridge, 2003
) (DNQX, 25.2 ± 9.5 s SEM, vs vehicle, 1.9 ± 0.5 s SEM; F(1,13)
= 6.533, p
= 0.024) (-). The amount of defensive treading was also 10 times greater after DNQX in caudal sites than in rostral sites in medial shell (DNQX, 0.18 ± 0.13 s SEM; vehicle, 0.53 ± 0.32 s SEM; caudal vs rostral; F(1,22)
= 4.698, p
= 0.042). Caudal stimulation of defensive treading behavior by DNQX appeared especially potent at ventral sites in the caudal half of medial shell, compared with more dorsal sites ().
Dopamine antagonists prevent caudal elicitation of fear
Adding the dopamine D1 and D2 antagonists to the DNQX microinjection blocked all increases in defensive treading behavior normally produced by DNQX alone at local sites in caudal shell (F(1,13) = 5.044; p = 0.043), just as dopamine blockade had blocked appetitive behavior in rostral shell (-). Even at far caudal sites in medial shell, microinjections of the combined antagonist mixture in the nucleus accumbens shell produced no more defensive treading behavior than vehicle microinjections (F(1,13) = 3.759; p = 0.075). Thus, there was no rostrocaudal difference in defensive treading performance after mixture microinjection (F(1,22) = 1.902; p = 0.182), no increase over vehicle control levels (F(1,21) = 3.885; p = 0.062), and no interactions between drug and sites (F(1,21) = 0.837; p = 0.371). Further results from the additional control group indicated, similar to eating results, that dopamine antagonists by themselves in the absence of DNQX did not alter spontaneous defensive treading behavior significantly from vehicle levels at any site, whether in rostral shell (F(1,6) = 2.356, ns) or caudal shell (F(1,4) = 1.402, ns) ().
DNQX at caudal sites also induced locomotion, such as crossing of grid squares in the chamber (F(1,13) = 9.701; p = 0.008), but not at rostral sites (F(1,8) < 1, ns). Conversely, DNQX at rostral sites suppressed rearing behavior (F(1,8) = 5.858; p = 0.042). DNQX had no effects on other general activity behaviors measured or on drinking (F values < 1). Addition of DA antagonists blocked the DNQX-increase in locomotor crossing (F(1,13) = 8.834; p = 0.011). However, addition of DA antagonists to DNQX did not reduce motor locomotion crossing (F < 1) or burrowing behavior (F(1,21) = 2.2, ns) below vehicle control levels, again consistent with the idea that dopamine antagonist microinjections did not simply make rats unable to engage in behaviors.
Anatomical control sites
Outside of the medial shell, at control sites dorsal or rostral to shell in cingulate cortex, lateral septal nucleus, or near the ventral tip of the corpus callosum (dorsal tenia tectum and navicular nucleus) (Paxinos and Watson, 2004
), DNQX produced no changes in either defensive behavior (F
< 1) or appetitive behavior (food intake, F(1,5)
= 1.29, ns; eating duration, F
< 1; drinking intake, F(1,5)
= 2.73, ns). Similarly, DNQX at control sites had no effect on other motor behaviors measured here (locomotor grid crossing, F(1,5)
= 3; burrowing and grooming, F(1,5)
= 4.31, ns).