Given the novel nature of the technique, we first wanted to validate the temporal sensitivity by examining time-dependent brain activity changes in a well-characterized neuronal circuit that could be directly studied in relation to exogenous stimulation. Therefore, we used the well-established vibrissae stimulation model, which is known to activate the barrel field somatosensory cortex (11
). We scanned naive adult (P55-P69) male Sprague Dawley (SD) rats (n
= 6) using a dynamic scanning procedure in which anesthetized rats were subjected to continuous unilateral vibrissae stimulation that was initiated 5 minutes prior to intravenous i.v. FDG injection. Scans were then normalized to Paxinos stereotaxic coordinates (12
) and analyzed using statistical parametric mapping (SPM) as previously described (7
). Using this dynamic approach, we observed time-dependent contralateral barrel field circuit activation as early as 1 minute after FDG injection (Figure ). The anatomical specificity of this activation directly overlapped known connectivity of the barrel field circuit (ref. 11
and Figure ), exemplifying the sensitivity of our imaging methodology in detecting functional changes in brain activity with significant anatomical and temporal specificity (single-minute resolution). To our knowledge, this is the first report documenting the ability to detect time-dependent changes in FDG brain uptake at this temporal and spatial resolution.
Vibrissae stimulation leads to time-dependent brain activation in barrel field circuitry.
For DREAMM experiments, we used herpes simplex virus (HSV) vectors expressing an engineered DREADD receptor under the control of the Pdyn
promoter, which had been previously validated to drive selective expression of the inhibitory Gi
Di DREADD in Pdyn-
) and whose specificity we also validated here (Figure , A and B). This strategy would thus allow, respectively, Gi
-mediated inhibition of “Go,” or activation of “NoGo” MSN output pathways (10
). First, we assessed whether hM4
Di-mediated inhibition of NAcSh Pdyn-
MSNs reduced neuronal activity locally. Adult (P55–P69) male SD rats were infused into the right NAcSh with the Pdyn
- or Penk
Di HSV constructs (n
= 6/group) (Figure C). Two weeks later, rats received an i.p. injection of clozapine-n-oxide (CNO) (1 mg/kg), which promotes Gi
-coupled inhibition of hM4
), were sacrificed 1 hour later and processed for c-Fos immunohistochemistry. Consistent with prior reports in which Gi
-mediated activation was associated with decreases in c-Fos (13
), we found that hM4
Di activation in NAcSh Pdyn-
MSNs decreased c-Fos activation locally in the ipsilateral NAc as well as in the VTA (Figure ). On the other hand, Penk-
MSN inhibition decreased c-Fos activity only in the ipsilateral NAc, but not in VTA (Figure ). These findings emphasize the specificity in neuronal inhibition achieved using DREADD.
Validation of HSV vector selectivity and infusion sites.
Cell-type–specific control of local and long-range signaling is detected using c-Fos immunostaining.
Next, we attempted to detect in vivo the functional whole-brain dynamic circuits associated with inhibition of Pdyn-
MSNs. Male adult SD rats (n
= 6/group) were infused into the right NAcSh with Pdyn
- or Penk
Di constructs and scanned at 7 (after i.p. injection of vehicle) and 14 days (after i.p. CNO) after vector injection. Immediately after injection (vehicle or CNO), rats were anesthetized (1.5% isoflurane), injected i.v. with FDG (~0.6 mCi) and scanned using the above dynamic scanning methodology. All images were then normalized to Paxinos stereotaxic coordinates (12
) and analyzed as previously described (7
). Significant DREAMM responses in areas with direct, known connectivity of Pdyn-
MSNs were observed as early as 1 minute after scanning (Figure ). In particular, Pdyn
-MSN inhibition led to a significant increase in FDG uptake in areas coincident with the posterior VP and VTA/substantia nigra (SN) (Figure A), whereas Penk
-MSN inhibition led to a significant decrease in FDG uptake in a cluster approximating the rostral VP (Figure B). Similar to the vibrissae stimulation model in which we observed time-dependent recruitment of barrel field circuits, Pdyn-
MSN inhibition resulted in discrete time-dependent DREAMM responses that spanned recruitment of specific brain regions and revealed distinct network patterns for each MSN subtype (Figure , A and B). In particular, Pdyn
-MSN inhibition induced a time-dependent continuum of decreased FDG uptake throughout the ipsilateral ventral forebrain extending from the olfactory tubercle through the hypothalamus to the interpeduncular midbrain region and brainstem. This ventral inhibitory DREAMM circuit was coincident with a broad time-dependent increase in FDG uptake in the dorsal pallidum and midbrain followed by other dorsal structures extending from midline cingulate cortices (CG), throughout the dorsal hippocampus (HP) to the cerebellar nuclei (Figure A). In contrast, Penk
-MSN inhibition was predominantly characterized by a time-dependent reduction of ipsilateral FDG uptake in the olfactory tubercle, VP, globus pallidus, ventral pons, sensory brainstem nuclei, and cerebellum as well as ipsilateral FDG increases in the dorsal HP, caudate putamen, and septofimbrial nucleus (Figure B). The more widespread effects of Pdyn
-MSN inhibition as compared with Penk
-MSN implicates a greater role of the “Go” pathway in modulating brain activity under anesthetized conditions.
Cell-type–specific, time-dependent control of local and long range signaling is detected using DREAMM in anesthetized rats.
To validate the specificity of DREAMM to discern FDG changes within discrete brain regions, nonbiased SPM results were also expressed quantitatively using a novel region of interest–based (ROI-based) image analysis method that used Paxinos stereotaxic rat coordinates to extract individual subject values for the VTA/SN. Using this approach, individual subject values plotted for the VTA/SN, which specifically dissociate Pdyn
projections, revealed that, in agreement with SPM, inhibition of Pdyn-
MSNs led to a significant increase (8.6%) in FDG uptake in the VTA/SN at 1 minute into the scan, whereas after 30 minutes, and consistent with both SPM and c-Fos results, CNO significantly decreased FDG uptake (7.2%) in this region (Figure C). Importantly, in agreement with a lack of direct anatomical connectivity with the VTA/SN, no significant differences were observed in this region at either 1 or 30 minutes in response to Penk
-MSN inhibition (Figure D). Many studies have now shown convincingly that CNO does not have any off-target effects in a variety of experimental parameters in both rats and mice (13
). In order to expand this to brain and peripheral glucose metabolism, and since the transaxial field of view of the scanner did not accommodate whole-body rat scans, we scanned naive adult male mice (n
= 6) twice, once after i.p. CNO (1 mg/kg) and once after i.p. vehicle injection. Consistent with numerous prior observations for a lack of off-target effects of CNO on behavior, neurophysiology, and pharmacology in both rats and mice, we did not observe any significant changes in FDG uptake between vehicle and CNO scans (Supplemental Figure 1; supplemental material available online with this article; doi:
). Overall, these results suggest that, in anesthetized rats, DREAMM is able to successfully capture cell-specific, time-dependent changes in brain functional activity with single-minute resolution.
Next, we assessed DREAMM functional connectivity of NAcSh Pdyn-
-MSNs associated with FDG uptake in the awake state using a well-established behavioral imaging paradigm (6
). Male adult SD rats (n
= 6) were injected i.p. with vehicle or CNO and placed in an open-field arena. At 30 minutes, rats were injected i.p. with FDG (~0.6 mCi), and 30 minutes subsequently, they were anesthetized and scanned for 20 minutes using a static acquisition protocol. We found that reducing neuronal activity of the right NAcSh Pdyn
-MSNs significantly increased left turn motor behavior (P
= 0.006) (Figure A), and this was paralleled by significant increases in counter-clockwise (leftward) circling (P
= 0.001) (Figure B). Interestingly, Pdyn
-MSN inhibition significantly increased (8.8%) FDG uptake in the right motor cortex (MC) (Figure C). Inhibition of Penk
-MSNs significantly decreased FDG uptake in MC bilaterally, though this was more pronounced in the right MC (14.4%) (Figure F). In line with the DREAMM pattern, CNO increased ipsilateral (right) versus contralateral turns (P
= 0.05) (Figure D), but there was no difference in general clockwise vs. counter-clockwise circling (Figure E). These findings are consistent with contralateral control of motor behavior by the MC and emphasize the ability of DREAMM to map behavior to global brain functional circuits of discrete yet spatially overlapping cell types. Paradoxically, the directionality in turning behaviors we currently observed after inhibition of direct and indirect pathway ventral striatal MSNs was similar to that previously reported by Kravitz et al. after activation of the dorsal MSN pathways (21
). Interestingly, duration of stimulation may account for these findings, since acute and sustained inhibition led to time-dependent, opposing changes in FDG uptake in downstream projection sites. Such time-dependent changes in FDG uptake are likely mediated by changes in complex downstream signaling plasticity mechanisms relevant to spatiotemporal control of Gi
Unilateral inhibition of Pdyn- and Penk-expressing neurons in NAcSh leads to opposing patterns in turning behavior and MC activity.
Similar to what was found under anesthetized conditions, discrete DREAMM responses were observed following Pdyn-
-MSN inhibition in freely moving rats that had distinct limbic-related profiles associated with each NAc pathway (Figure ). Pdyn
-MSN inhibition was associated with significant FDG uptake in ipsilateral corticolimbic circuits, including a striking 17.8% ipsilateral increase in the medial entorhinal cortex (Ent) and approximately 10%–12% alteration throughout the ipsilateral rostrocaudal extent of the VP and horizontal limb of the diagonal band (HDB) (Figure , A and B). There were also significant bilateral increases in medial amygdala (MEA) (13% ipsilateral; 4.8% contralateral) and retrosplenial cortex (12.3% ipsilateral; 9.8% contralateral). In addition, FDG uptake was increased in the entire contralateral rostrocaudal extent of the dysgranular insula (DI) (8.1%) and CG (8.2%). Overall, only increased FDG uptake was observed with NAcSh Pdyn
-MSN inhibition, whereas both increased and decreased uptake were observed with Penk
-MSN inhibition in the awake, moving rodent (Figure , C and D). Intriguingly, inhibiting Penk
-MSN activity resulted in significant ipsilateral FDG increases in primary components of the limbic system including the VP (8.8%), amygdala (basal [14%] and medial [8.3%] divisions), and HP (8.6%) (pronounced demarcation of the dentate gyrus). Such HP and amygdala activation is particularly interesting, given recent studies demonstrating a role for the “indirect” NAcSh Penk
-MSNs in mediating behavioral responses associated with aversive memory formation (24
). Indeed, a unique feature of the Penk
-MSN manipulation was increased FDG uptake (8.2%–11.3%) along the fornix, which primarily connects the HP with the septum and diencephalon. Interestingly, NAcSh Penk
-MSN inhibition also led to a striking bilateral activation of the DI (14.6% ipsilateral and 17.3% contralateral), and ventromedial PFC, particularly the medial orbital area (12.4% ipsilateral and 12.5% contralateral). Finally, and in contrast to Pdyn
-MSN inhibition, Penk-
MSN inhibition led to significant decreases in FDG uptake in the ipsilateral sensory cortex (SC) (14.7%), globus pallidus (GP) (5.7%), and contralateral piriform cortex (Pir) (17.6%). Overall, these findings identify distinct neural activity associated with Pdyn
limbic striatal pathways and emphasize that direct NAc pathway impairments have profound bottom-up regulation, particularly of limbic-related cortices. Additionally, the recruitment of motor systems (e.g., dorsal striatopallidal circuit and MC) by NAcSh Penk
-MSN inhibition emphasizes the strong limbic-motor interface of this pathway.
Unilateral inhibition of spatially overlapping Pdyn- and Penk-expressing neurons in NAcSh of freely moving rats leads to changes in distinct neural circuits.