Previously, we demonstrated that [11
C]-salvinorin A, given intravenously in non-human primates, rapidly enters the brain, distributes in a high concentration to the cerebellum and throughout the cortex, but persists in the brain for only minutes [29
]. Indeed, we found that a similar distribution occurred in the rat brain with even faster kinetics such that binding was difficult to discriminate from blood flow (time to peak, 20 sec; half life from peak, 180 sec). Clearly, the pharmacological duration of action of SA and its unique abuse liability as a KOR agonist directly correlate with its kinetics, but so far the kinetics and distribution provide limited insight into the behavioral effects seen in rodents.
To examine the effects of SA on regional brain function, we investigated regional brain metabolic changes in rodents given an acute dose of SA (2.0 mg/kg i.p. in DMSO). Control animals were given vehicle injections of DMSO using an identical protocol. By imaging the rodent brain with small animal positron emission tomography (mPET) after administration of SA and 18FDG, regional drug-dependent differences were determined. Patterns of change in 18FDG uptake associated with drug administration give a measure of the downstream effects of drug action. Regional patterns of activation and deactivation in response to an acute drug challenge can be used to identify a metabolic `signature' comprised of relevant regions of the brain for different compounds within the same chemical class, or different chemical classes. Many SA behavioral paradigms in the literature have pointed to depression, antinociception, and decreased locomotion as prominent effects of SA administration. We anticipate that combining these behavioral observations with information about discrete metabolic changes in the rat brain when exposed to SA (reported herein), will offer a more complete understanding of the pharmacological properties of this potent and selective KOR agonist.
Our experimental design was guided by behavioral and physiological data in the literature [16
]. In rodents, SA (i.p.
) quite consistently reached its maximum effect between 20 and 40 min, as measured through behavioral challenge or dopamine change measure via
microdialysis. Thus, we designed our experimental protocol to allow for maximal 18
FDG uptake during this period, . After administering SA (2.0 mg/kg i.p.
) or vehicle only (0.1 mL DMSO), each animal was returned to its home-cage for 12 min. Following this, 18
FDG (nominally 1.0 mCi, i.p.
) was given. 18
FDG administered in this manner reaches a maximum concentration in the brain in 10-15 min, which corresponds to 22-27 min post administration of SA [27
]. Thus, using this timeline, the peak of 18
FDG uptake into the brain was timed to coincide with of that of SA.
Animals were allowed to behave freely until 1 hr post SA administration at which time they were anesthetized with ketamine/xylazine. Each animal's locomotion was recorded during the 18
FDG uptake period (i.e. 12-60 min post SA injection). Analysis of locomotion data indicated no statistically significant difference between the two groups of animals (all analyzed time bins, P
> 0.3). While a single acute dose did not have an effect on locomotion, in subsequent experiments we observed locomotion changes after multiple acute doses (see electronic supplementary material
After imaging and data processing (see details in methods), groups of images (SA, n = 9; vehicle, n = 10) were compared using statistical parametric mapping (SPM). Using a statistical tolerance of P
< 0.05, t-map images were generated and superimposed on a MRI template of the rat [28
], all in stereotaxic space. Representative two-dimensional images from this three-dimensional data set are shown in .
Regional differences in functional brain activity in rats given SA (n = 10) compared to vehicle-treated controls (n = 9)
The data were interrogated at the cluster level and assigned to a structural region. The percent change (either increase or decrease) in 18FDG uptake for the group of SA images relative to DMSO controls was determined using a spherical region of interest of 2.5 mm diameter surrounding the voxel of greatest intensity (i.e. highest statistical significance) within each cluster. This ensured minimal overlap in cluster analysis. The results of SPM and subsequent analysis are summarized in .
Summary of cluster- and voxel-level statistics determined with SPM
All of the data showed striking symmetric bilaterally suggesting a high degree of fidelity. The absolute magnitude of change may be masked by using a whole brain normalization, which eliminates differences in absolute 18FDG uptake, but nonetheless the regional interrogation and results can be used for hypothesis driven behavioral research on SA and its derivatives in the future.
As might be expected, several of regions with high KOR density did indeed show increased 18
FDG uptake. Of particular note, the periaqueductal gray (PAG), showed increased metabolic activity (, white arrow). This cerebral duct within the midbrain has a very high density of KOR and has been repeatedly correlated with modulation of pain, as well as in defensive behavior and fear conditioning [30
]. Activation of KORs in the dorsal PAG has also been linked with defensive behavior in rats tested in the elevated plus maze [31
]. We also observed significant bilateral activation of the bed nucleus of the stria terminalis (BNST; , orange arrows), a region with high density of KOR (Mansour et al., 1996). In addition, the vermis of the cerebellum was highly activated. This region has recently been ascribed the function of proprioception in animals although it is unclear whether activation would impair or enhance spatial awareness [32
Regional differences in 18FDG uptake were not limited to brain regions associated with a high density of KOR. Significant bilateral activations were also observed in regions which have little or no KORs, such as the hypothalamus, auditory, sensory and frontal cortices (). Relative increases in 18FDG uptake were also observed unilaterally in the left ventral pallidum and right lateral geniculate nuclei. These regional differences may reflect neuronal activity downstream from the changes in neural activity at sites with high KOR density, as well as the subsequent recruitment of additional brain regions.
Bilateral metabolic decreases were observed in the caudate putamen, superior colliculus, hippocampus, and medial brainstem (, blue regions). These results show that the metabolic response to SA goes well beyond the immediate KOR effects and involve a larger activation of neuronal circuits projecting from the primary KOR sites to functionally and anatomically related regions of the brain. For instance, decreases in metabolism may result from activation of inhibitory neurons projecting to these regions. With only a map of relative local glucose utilization, distinguishing between changes resulting from local cellular interactions and those that are interneuron mediated is not possible. For a review on the role of inhibitory interneurons on the brain's energy consumptions, see Buzsáki et. al. [33
Here we show localized changes in brain activity resulting from an acute challenge of SA that are specific and extend beyond its initial site of action. Although we can only speculate at this point on the relationship of activation or deactivation of particular brain regions to behavior, we feel these data may provide a basis for interrogating the effects of SA in rodents in the future. Identification of brain regions demonstrating changes in neural activation in response to SA may inform the design of future analogs of SA and may provide an experimental platform for simultaneous testing of the pharmacodynamic and behavioral effects of these new compounds in the same animal. For example, structural manipulations of SA, in vitro
structure-activity relationships, and an understanding of molecular interactions can lead to more potent and longer lasting drugs [34
]. The impact of these developments, both physiologically and behaviorally, can be marked [38
]. We feel there will be an increasing need to systematically understand how affinity, pharmacokinetics, and distribution influence behavior through regional changes in brain activation. Our studies highlight the potential of 18
FDG with small animal PET to accomplish this.
Finally, it is worth noting that the route of SA administration (ip
) may have a prevailing impact on pharmacodynamics and behavior in animals as noted in the human experience [39
]. We are currently investigating how the route of administration (and therefore SA pharmacokinetics) effects brain glucose utilization as well as probing the effects of other SA derivatives that exhibit longer last effects.