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Fatty acid amide hydrolase (FAAH) activity is known to mediate the tone of endogenous fatty acid amides including the endocannabinoid anandamide (AEA). FAAH is a potential therapeutic target becuase genetic or pharmacological ablation of FAAH promotes analgesia and anxiolytic effects without disrupting motor coordination. Little is known about the endogenous temporal fluctuations of brain FAAH activity. This is the first comprehensive study examining temporal fluctuations in mouse brain FAAH activity. Regional mouse brain homogenates were generated at the midpoint of the light (“noon”) and dark (“midnight”) cycles. While immunoblots revealed no significant change (P>0.05) in regional activity between these two time points, in vitro activity assays detected a subtle 10% reduction (P<0.05) in cerebellar FAAH activity at midnight. A novel ex vivo autoradiography technique permitted the study of eleven different brain regions, many of which cannot be studied using traditional in vitro methods. The cerebellum and the PAG both exhibited significant (P<0.05) reductions in regional FAAH activity in ‘midnight’ brains. These data confirm the need to account for temporal changes in FAAH activity when therapeutically targeting FAAH.
The endocannabinoid system mediates numerous physiological processes, including reproduction 12, metabolism 3, immunity 36, and digestion 1. Through the activation of cannabinoid receptor 1 (CB1R), endocannabinoids play a central role in CNS-mediated functions including nociception 17, motor coordination 11, memory 11, and vision 42. Signaling of the endocannabinoid arachidonoyl ethanolamide (AEA) is terminated by its hydrolysis by fatty acid amide hydrolase (FAAH). Because FAAH knockout mice posses elevated endogenous fatty acid amide levels and are unable to efficiently metabolize exogenously administered AEA 4, FAAH is the principal enzyme mediating the in vivo metabolism of AEA other fatty acid amides (for review, see 25). FAAH also hydrolyzes the endocannabinoid 2-arachidonoylglycerol (2-AG) in vitro 15, although its contribution toward 2-AG inactivation in vivo is currently not known.
As expected, CB1R and FAAH are distributed throughout the mouse brain, with robust immunoreactivity in the hippocampus and dorsal tenia tecta. Additional FAAH immunoreactivity occurs in the olfactory bulb, cerebral cortex, thalamus, and cerebellum, predominantly in dendrites and somata post-synaptic to CB1R immunoreactive axons 9. This expansive immunoreactivity pattern suggests that FAAH plays a critical role in mediating AEA signaling in the brain.
Selective FAAH inhibitors produce a subset of the behavioral effects observed with CB1R agonists. For example, i.p. administration of URB597 increased endogenous AEA levels and produced analgesia and anxiolytic effects, without promoting catalepsy, hypothermia, or hypophagia in rats 19. Similarly, administration of the selective FAAH inhibitor OL-135 produced analgesic effects in mice as determined by hotplate and tail immersion tests 22. Therefore, FAAH activity mediates endocannabinoid tone in brain regions responsible for pain and anxiety. Cannabinoid-mediated effects such as catalepsy, hypothermia, and hypophagia are likely regulated by enzymes other than FAAH, as these behaviors were unaffected by inhibitor administration.
While FAAH is principally responsible for AEA inactivation, several other enzymes are also capable of metabolizing AEA. Cyclooxygenase-2 metabolizes AEA in in vitro assays 10, 43 and its activity limits endocannabinoid signaling in the hippocampus 21. FAAH-2 39 and N-acylethanolamine-hydrolyzing acid amidase 35 hydrolyze AEA, although they are more effective in metabolizing oleamide and palmitoylethanolamide, respectively. The in vivo contributions of FAAH-2 and NAE-hydrolyzing acid amidase remain unknown, and their CNS contributions are likely minimal. NAE-hydrolyzing acid amidase is expressed primarily in the periphery (lungs, spleen, small intestine, thymus and immune cells) 35, and may mediate fatty acid amide inactivation in these tissues. In primates and several other species, FAAH-2 is widely expressed in peripheral tissues including kidney, liver, lung, prostate, heart, and ovary. It is important to note that FAAH-2 has not been found in the brain of any species examined, nor is it expressed in mice or rats 39.
Very few studies have examined temporal changes in endocannabinoid levels and their metabolism. With respect to FAAH, Valenti et al. determined regional endogenous AEA levels increased, and rat brain FAAH activity decreased, at midnight relative to midday brain samples 37. These data are complemented by a second study indicating oleamide, palmitoylethanolamide, and AEA levels are highest in rat cerebrospinal fluid during the “lights-off” period 27. Epithelial cells of the rat choroid plexus, a region known to produce cerebrospinal fluid, are FAAH-IR 8, suggesting FAAH activity in these cells may also vary temporally. Mounting evidence indicates that AEA modulates sleep (for review, see 26), so temporal changes in regional FAAH activity may also affect sleep patterns.
In this manuscript, we utilized a novel ex vivo autoradiography technique to examine temporal changes in regional FAAH activity (See 14 for a detailed characterization of this assay). Unlike in vitro homogenate assays and RT-PCR, this novel autoradiography technique takes into account both FAAH activity levels and the capacity of AEA trafficking mechanisms such as FABPs18, as FAAH activity is measured following substrate delivery in intact tissue. In addition, brain regions impossible to accurately dissect, and regions too small for homogenate generation, can easily be studied. Temporal changes in FAAH activity were examined throughout mouse brain. Mice were intravenously administered [3H]AEA at the midpoint of the light (“noon”) or dark (“midnight”) cycle. Differences in regional tritium distribution patterns, representing changes in regional [3H]AEA hydrolysis capacity between these two time points, were determined. Data from these autoradiography studies were compared to traditional in vitro homogenate assay and immunoblot data to confirm the results and techniques of this study.
C57BL/6 mice (Taconic Farms, Germantown, NY) weighing approximately 25g were kept in LD 12:12. noon mice were studied at the midpoint of the light cycle, while midnight mice were studied at the midpoint of the dark cycle. All mice were kept in the same room and were maintained with free access to food and water according to the federal guidelines for the care and use of animals. These studies were approved by the Institutional Review Committee.
Six hours after the onset of the light and dark period, regional brain dissections were performed on a chilled surface. Following tissue homogenization on ice in Tris + 1mM ethylenediaminetetraacetic acid (EDTA) + complete Mini EDTA acid -free protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany), protein concentrations were determined by BCA protein assay (Pierce Chemical, Rockford, IL). 40 μg of protein/lane were run on a 10% SDS-PAGE gel. Following transfer to a nitrocellulose membrane at 100V for 25 min, blots were blocked for one hour in 5% non-fat dry milk in PBS Tween (PBST). The blots were probed for one hour while shaking with polyclonal rabbit FAAH antibody directed against aa 561–579 of rat FAAH (Alexis Biochemicals, San Diego, CA) at a final concentration of 1:250 or monoclonal mouse anti-β actin antibody directed against aa 1–14 of Xenopus laevis actin (AbCam, Inc. Cambridge, MA) at a final concentration of 1:10,000. The blots were rinsed three times with PBST followed by incubation with goat anti-mouse or goat anti-rabbit IgG HRP-conjugated antibodies (Molecular Probes, Eugene, OR) for 1 hour. The blots were rinsed three times with PBST, developed using the Immun-star HRP substrate (Bio-Rad, Hercules, CA) and exposed to film. To determine changes in FAAH protein levels between noon and midnight samples, FAAH levels were normalized to actin levels within the same lane, and analyzed by densitometry using Image J software. Significance was determined by paired 2-Tailed T-test using Excel (Microsoft).
Six hours after the onset of the light and dark period, regional brain dissections were performed. Following tissue homogenization in Tris + 1mM EDTA on ice, protein concentrations were determined by BCA protein assay (Pierce Chemical, Rockford, IL). FAAH activity assays were performed according to published methods6. Briefly, 300 μg of regional homogenate, 500μg fatty acid-free bovine serum albumin, 100 μM AEA (Cayman Chemical, Ann Arbor, MI) plus 0.22 μCi arachidonoyl ethanolamide [ethanolamine-1-3H] (a gift from the National Institute on Drug Abuse) were incubated in Tris + 1mM EDTA pH 7.6 at 37°C in triplicate for fifteen minutes while shaking. The reactions were terminated by the addition of 2 volumes of 1:1 chloroform/methanol. Samples were centrifuged, and the aqueous phase measured by liquid scintillation counting. The rates of AEA hydrolysis/mg protein/hr were compared between noon and midnight regional homogenates. Significance was determined by paired 2-Tailed T-test using Excel (Microsoft). Data were graphed using GraphPad Prism 4 (GraphPad Software Inc., San Diego, CA).
Radiolabeled AEA [arachidonoyl 5,6,8,9,11,12,14,15-3 H] was a generous gift from the NIDA Drug Inventory Supply Program. AEA (Cayman Chemical) and [3H]AEA were dissolved in a 1:1:18 mixture of ethanol: emulphor: saline. Mice were i.v administered 1 mg/kg AEA + 2 mCi/kg [3H]AEA. Fifteen minutes following injection, mice were sacrificed and their brains promptly transferred to a beaker containing iced saline, in order to minimize further diffusion and protein-mediated processes. Half of the cerebellum and a blood sample were homogenized, extracted in 1:1 chloroform:methanol, and analyzed by scintillation counting and thin layer chromatography (solvent system was 6:3:1 ethylacetate:hexane: acetic acid6), in order to quantify total tritium accumulation, and [3H]AEA breakdown in these tissues. Chilled brains were transferred into iced 2% formaldehyde + 2% glutaraldehyde in PB for one hour. The brains were then washed three times with iced PB, and cryoprotected at 4°C in 30% sucrose in PB. Serial 15μm cryosections were generated, and images acquired for ten hours using the BetaImager (Biospace, Paris, France).
Serial sections were analyzed, and regions of interested identified using a commercially available mouse brain atlas26 as a reference. Surface radioactivity (cpm/mm2) in regions of interest was quantified using Beta Vision image analysis software (Biospace Mesures, France). Data were entered into Excel (Microsoft Corp., Seattle, WA), and average regional tritium accumulation/mm2, representing AEA and its metabolites, was determined. Six mice were used for each condition. Regional surface activities of each mouse were normalized by dividing the activity of each region over that of the pontine nuclei (the region of lowest tritium accumulation) of the same mouse. Normalization enabled regional comparison among animals with slightly different levels of brain tritium uptake. Standard error was calculated using Excel. Data were graphed, and one-way analysis of variance with Dunnett’s post test was performed to determine significance between noon and midnight samples using GraphPad Prism 4 (Graph Pad Software Inc., San Diego, CA).
To examine regional changes in FAAH protein levels, regional brain dissections were performed at the midpoint of the light (“noon”) and dark (“midnight”) cycles, and regional homogenates generated. First, immunoblots were run (Fig. 1) and densitometry analyses of FAAH protein levels were performed following normalization to β-actin (Fig. 2). Immunoblots detected no significant (P>0.05) changes in normalized FAAH protein levels in the cortex, hippocampus, cerebellum, striatum, or thalamus.
While changes in protein levels were not detectable, it remained possible that FAAH activity levels differed between noon and midnight. Therefore, we incubated regional homogenates with [3H]anandamide and compared the in vitro generation of [3H]ethanolamine by noon and midnight samples (Fig. 3). No significant difference (P>0.05) was observed between noon and midnight cortical, hippocampal, striatial, and thalamic homogenates. However, a subtle, yet significant (P<0.05) decline in cerebellar FAAH activity was detected at midnight relative to noon samples.
Regional FAAH activity was next analyzed by ex vivo imaging. For ex vivo imaging, mice were administered [3H]AEA+1mg/kgAEA at the midpoint of either the light (“noon”)or dark (“midnight”) cycle, and their brains isolated and fixed fifteen minutes later. Sections were generated and imaged in the BetaImager. Following tracer administration, [3H]AEA circulates through the blood and is either metabolized in the periphery or passes the blood brain barrier and enters the brain. As described in the trapping mechanism schematic (Fig. 4), [3H]AEA is subsequently taken-up by cells with FAAH and metabolized into [3H]arachidonic acid ([3H]AA). This is very similar to FAAH activity driving [3H]AEA uptake into cultured cells 5, 7. [3H]AA is subsequently incorporated into the membranes of the same cells that hydrolyzed the tracer. As a result, cells with the highest levels of [3H]AEA hydrolysis trap the most tritium in their membranes. In sharp contrast to WT mice, radiotracer administered to FAAH knockout mice remains unmetabolized. The intact [3H]AEA distributes in a homogeneous and diffuse manner 14, becuase the radiolabel escapes membrane entrapment.
Ex vivo imaging is more sensitive technique than in vitro FAAH activity assays because use of the latter is limited to regions of a minimal size that one can efficiently isolated by dissection. In addition, becuase tissue is not disrupted during AEA hydrolysis, ex vivo imaging permits both the determination of the tissue’s capacity for AEA hydrolysis by FAAH and the capacity of AEA delivery mechanisms such as fatty acid binding proteins18. Because substrate delivery to FAAH affects the rate of AEA hydrolysis, ex vivo imaging provides a more accurate representation of regional FAAH capacity than in vitro activity assays. We utilized this technique to compare FAAH activity levels in eleven regions of noon and midnight brains (Fig. 5).
Similar to the in vitro FAAH activity data, tritium accumulation in most brain regions did not significantly vary (P>0.05) between noon and midnight brains (Fig. 6). In addition, ex vivo imaging confirmed a slight decrease (P<0.05) in cerebellar FAAH activity in midnight brains relative to noon samples. Ex vivo imaging also detected a significant decline in FAAH activity in the periaqueductal gray (PAG), a region too small to analyze by traditional in vitro methods.
Analysis of brain samples by thin layer chromatography (run in parallel to these imaging experiments) indicated the bulk of tritium in these mouse brains was in the form of [3H] phospholipids (69% +/−5%). This is consistent with our prior publication (Glaser et al. 2006 JPET 316:1088–1097) and suggests the majority of tritium was trapped in regional membranes following [3H]AEA hydrolysis by FAAH.
There has been little investigation of temporal changes of enzymes mediating endogenous AEA tone. Valenti et al. reported an increase in endogenous AEA levels in the nucleus acumbens, prefrontal cortex, striatum, and hippocampus of the rat brain in the dark phase. This increase in endogenous AEA tone complemented a decline in FAAH activity in the striatum and hippocampus 37. In further support of these observations, endogenous levels of the FAAH substrates AEA, palmitoylethanolamide, and oleoylethanolamide have been shown to vary with a circadian cycle in several rat brain regions and in cerebrospinal fluid 27.
This is the first comprehensive study of temporal changes in regional FAAH activity in the mouse brain. The novel imaging technique14 permitted the examination of brain regions too small to analyze by standard in vitro methods, thereby producing the most thorough characterization of regional temporal fluctuations in FAAH activity to date. In sharp contrast to prior rat studies37, both regional and temporal variations in mouse brain FAAH activity are more subtle, with only the mouse cerebellum and PAG exhibiting significantly reduced tritium accumulation in midnight brains (Fig. 6). No nighttime increases in tritium accumulation were observed in any brain region. In vitro FAAH activity assays confirmed that midnight cerebellar homogenates exhibited slightly reduced in vitro activity relative to noon samples (Fig. 3). Importantly, these experiments demonstrate the sensitivity of this novel autoradiography technique, and provide proof that similar results can be obtained with a traditional in vitro assay. As the difference in in vitro cerebellar FAAH activity was only 10%, it is not surprising that changes in protein levels were not detected by immunoblot (Fig. 2).
There is growing evidence that FAAH may represent an ideal pharmaceutical target for the design of novel analgesics. Unlike CB1R agonists that produce both beneficial effects and prominent psychotropic and motor effects, FAAH inhibitors provide analgesia and reduce anxiety without negative side-effects 19. As FAAH has great therapeutic potential for pain, temporal changes in PAG FAAH should be taken into account when optimizing treatment.
The observed temporal changes in mouse PAG FAAH activity support observations in prior stress-induced analgesia studies. Stress induced analgesia occurs when acute stress activates either opioid- or non-opioid signaling pathways 2, 38 in the brain that alleviate pain. While the opioid pathway is well studied 41, only recently were the endocannabinoids AEA and 2-arachidonoylglycerol shown to mediate the non-opioid component in the PAG. Non-opioid mediated stress-induced analgesia was potentiated by direct administration of inhibitors of endocannabinoid inactivation to the PAG and abolished by the CB1R antagonist Rimonabant 16, 33. Becuase the non-opioid-mediated component of stress-induced analgesia is known to exhibit a day-night rhythm with enhanced analgesia observed at night 20, it is expected that endocannabinoid levels would fluctuate with a temporal pattern within the PAG.
FAAH inhibition in the PAG following the microinjection of the selective FAAH inhibitor URB597 raises both endogenous AEA and 2-arachidonoylglycerol levels 24. Therefore, fluctuations in FAAH activity levels are sufficient to significantly alter endocannabinoid tone in this region. We observed [3H] accumulation levels (and therefore FAAH activity) significantly declined in the PAG of midnight brains relative to noon samples. The nocturnal decline in PAG FAAH activity observed in the current study may provide a molecular mechanism for the nocturnal increase of the non-opioid component of stress-induced analgesia.
The cerebellum is best known to mediate motor coordination. This brain region’s output, purkinje cells, are both FAAH and CB1R immunoreactive 9. Therefore, it was somewhat surprising that, unlike CB1R agonists, systemically administered FAAH inhibitors do not produce deficits in motor coordination 19, 22. It must be concluded that FAAH activity is not primarily responsible for regulating cerebellar endocannabinoid tone and motor coordination.
Becuase FAAH inhibition is insufficient to alter motor coordination in the cerebellum, the moderate decline (P<0.05) in in vitro FAAH activity and midnight cerebellar tritium accumulation (Figs 3 and and6)6) observed in this study is expected to have no negative effect upon motor coordination at night, when mice are most active. Instead, decreased cerebellar FAAH activity at midnight suggests FAAH is either mediating endocannabinoid hydrolysis remote from CB1R, or is mediating the tone of other substrates that have no effect upon CB1R stimulation in the cerebellum. It remains likely that either monoglyceride lipase alone, or FAAH and monoglyceride lipase in concert, mediate(s) endocannabinoid tone in the cerebellum 23, 34.
In conclusion, this is the first study to identify subtle temporal changes in regional mouse brain FAAH activity. Using traditional in vitro techniques, we determined FAAH activity in the cerebellum significantly declined in homogenates generated at midnight. Cortical, thalamic, and hippocampal homogenates exhibited no significant change in FAAH activity between noon and midnight samples. Using a novel autoradiography technique, we confirmed the significant reduction tritium accumulation (driven by [3H]AEA hydrolysis) in midnight cerebella following [3H]AEA administration. This sensitive method also permitted the identification of subtle reductions (P<0.05) in tritium accumulation in the PAG, a region too small to study by traditional in vitro methods. The temporal changes in PAG FAAH activity supports prior studies and suggests endocannabinoid involvement in circadian changes in non-opioid stress-induced analgesia. The observed changes in cerebellar FAAH activity are likely unrelated to any endocannabinoid-mediated declines in motor coordination at midnight in mice.
We would like to thank Dr. Stephen Yazulla for the use of his laboratory space and equipment while conducting this research. We thank Academic Press for the permission to reproduce images. We would like to acknowledge the NIH for their support of this study: NIH grant 1 K01 DA021806-01 (S.T.G.).
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