Dynamic neuroreceptor positron emission tomography in non-anesthetized rats using point source based motion correction: A feasibility study with [11C]ABP688.

To prevent motion artifacts in small animal positron emission tomography (PET), animals are routinely scanned under anesthesia or physical restraint. Both may potentially alter metabolism and neurochemistry. This study investigates the feasibility of fully awake acquisition and subsequent absolute quantification of dynamic brain PET data via pharmacokinetic modelling in moving rats using the glutamate 5 receptor radioligand [11C]ABP688 and point source based motion correction. Five male rats underwent three dynamic [11C]ABP688 PET scans: two test-retest awake PET scans and one scan under anesthesia for comparison. Specific radioligand binding was determined via the simplified reference tissue model (reference: cerebellum) and outcome parameters BPND and R1 were evaluated in terms of stability and reproducibility. Test-retest measurements in awake animals gave reliable results with high correlations of BPND (y = 1.08 × -0.2, r = 0.99, p < 0.01) and an acceptable variability (mean over all investigated regions 15.7 ± 2.4%). Regional [11C]ABP688 BPNDs under awake and anesthetized conditions were comparable although in awake scans, absolute radioactive peak uptakes were lower and relative blood flow in terms of R1 was higher. Awake small animal PET with absolute quantification of neuroreceptor availability is technically feasible and reproducible thereby providing a suitable alternative whenever effects of anesthesia are undesirable, e.g. in sleep research.

A genetic variation in the adenosine A2A receptor gene contributes to variability in oscillatory alpha power in wake and sleep EEG and A1 adenosine receptor availability in the human brain.

The EEG alpha rhythm (∼ 8-13 Hz) is one of the most salient human brain activity rhythms, modulated by the level of attention and vigilance and related to cerebral energy metabolism. Spectral power in the alpha range in wakefulness and sleep strongly varies among individuals based on genetic predisposition. Knowledge about the underlying genes is scarce, yet small studies indicated that the variant rs5751876 of the gene encoding A2A adenosine receptors (ADORA2A) may contribute to the inter-individual variation. The neuromodulator adenosine is directly linked to energy metabolism as product of adenosine tri-phosphate breakdown and acts as a sleep promoting molecule by activating A1 and A2A adenosine receptors. We performed sleep and positron emission tomography studies in 59 healthy carriers of different rs5751876 alleles, and quantified EEG oscillatory alpha power in wakefulness and sleep, as well as A1 adenosine receptor availability with 18F-CPFPX. Oscillatory alpha power was higher in homozygous C-allele carriers (n = 27, 11 females) compared to heterozygous and homozygous carriers of the T-allele (n(C/T) = 23, n(T/T) = 5, 13 females) (F(18,37) = 2.35, p = 0.014, Wilk's Λ = 0.487). Furthermore, a modulatory effect of ADORA2A genotype on A1 adenosine receptor binding potential was found across all considered brain regions (F(18,40) = 2.62, p = 0.006, Wilk's Λ = 0.459), which remained significant for circumscribed occipital region of calcarine fissures after correction for multiple comparisons. In female participants, a correlation between individual differences in oscillatory alpha power and A1 receptor availability was observed. In conclusion, we confirmed that a genetic variant of ADORA2A affects individual alpha power, while a direct modulatory effect via A1 adenosine receptors in females is suggested.

Effects of electroconvulsive therapy on cerebral A1 adenosine receptor availability: a PET study in patients suffering from treatment-resistant major depressive disorder.

Introduction: Sleep deprivation and electroconvulsive therapy (ECT) effectively ameliorate symptoms in major depressive disorder (MDD). In rodents, both are associated with an enhancement of cerebral adenosine levels, which in turn likely influence adenosinergic receptor expression. The aim of the current study was to investigate cerebral A1 adenosine receptor (A1AR) availability in patients with MDD as a potential mediating factor of antidepressant effects of ECT using [18F]CPFPX and positron emission tomography (PET). Methods: Regional A1AR availability was determined before and after a series of ECT applications (mean number ± SD 10.4 ± 1.2) in 14 subjects (4 males, mean age 49.5 ± 11.8 years). Clinical outcome, measured by neuropsychological testing, and ECT parameters were correlated with changes in A1AR availability. Results: ECT had a strong antidepressive effect (p < 0.01) while on average cerebral A1AR availability remained unaltered between pre-and post-ECT conditions (F = 0.65, p = 0.42, mean difference ± SD 3.93% ± 22.7%). There was no correlation between changes in clinical outcome parameters and regional A1AR availability, although individual patients showed striking bidirectional alterations of up to 30-40% in A1AR availability after ECT. Solely, for the mean seizure quality index of the applied ECTs a significant association with changes in A1AR availability was found (rs = -0.6, p = 0.02). Discussion: In the present study, therapeutically effective ECT treatment did not result in coherent changes of A1AR availability after a series of ECT treatments. These findings do not exclude a potential role for cerebral A1ARs in ECT, but shift attention to rather short-termed and adaptive mechanisms during ECT-related convulsive effects.

Correction of motion tracking errors for PET head rigid motion correction.

Objective. In positron emission tomography (PET) rigid motion correction, erroneous tracking information translates into reduced quality in motion corrected reconstructions. We aim to improve the accuracy of the motion tracking data, to improve the quality of motion corrected reconstructions.Approach. We developed a method for correction of marker/skin displacement over the skull, for tracking methods which require multiple markers attached on the subject head. Additionally, we correct for small magnitude (∼1-2 mm) residual translation tracking errors that can still be present after other corrections. We performed [18F]FDG scans in awake mice (n= 8) and rats (n= 8), and dynamic [18F]SynVesT-1 scans in awake mice (n= 8). Head tracking was performed with the point source tracking method, attaching 3-4 radioactive fiducial markers on the animals' heads. List-mode even-by-event motion correction reconstruction was performed using tracking data obtained from the point source tracking method (MC), tracking data corrected for marker displacement (MC-DC), and tracking data with additional correction for residual translation tracking errors (MC-DCT). Image contrast, and the image enhancement metric (IEM, with MC as reference) were calculated in these 3 reconstructions.Main results. In mice [18F]FDG scans, the contrast increased on average 3% from MC to MC-DC (IEM: 1.01), and 5% from MC to MC-DCT (IEM: 1.02). For mice [18F]SynVesT-1 scans the contrast increased 6% from MC to MC-DC (IEM: 1.03), and 7% from MC to MC-DCT (IEM: 1.05). In rat [18F]FDG scans contrast increased 5% (IEM: 1.04), and 9% (IEM: 1.05), respectively.Significance. The methods presented here serve to correct motion tracking errors in PET brain scans, which translates into improved image quality in motion corrected reconstructions.

Associations between resting state brain activity and A1 adenosine receptor availability in the healthy brain: Effects of acute sleep deprivation.

Introduction: Previous resting-state fMRI (Rs-fMRI) and positron emission tomography (PET) studies have shown that sleep deprivation (SD) affects both spontaneous brain activity and A1 adenosine receptor (A1AR) availability. Nevertheless, the hypothesis that the neuromodulatory adenosinergic system acts as regulator of the individual neuronal activity remains unexplored. Methods: Therefore, fourteen young men underwent Rs-fMRI, A1AR PET scans, and neuropsychological tests after 52 h of SD and after 14 h of recovery sleep. Results: Our findings suggested higher oscillations or regional homogeneity in multiple temporal and visual cortices, whereas decreased oscillations in cerebellum after sleep loss. At the same time, we found that connectivity strengths increased in sensorimotor areas and decreased in subcortical areas and cerebellum. Discussion: Moreover, negative correlations between A1AR availability and rs-fMRI metrics of BOLD activity in the left superior/middle temporal gyrus and left postcentral gyrus of the human brain provide new insights into the molecular basis of neuronal responses induced by high homeostatic sleep pressure.

Cerebral A1 adenosine receptor availability in female and male participants and its relationship to sleep.

The neuromodulator adenosine and its receptors are mediators of sleep-wake regulation which is known to differ between sexes. We, therefore, investigated sex differences in A1 adenosine receptor (A1AR) availability in healthy human subjects under well-rested conditions using [18F]CPFPX and positron emission tomography (PET). [18F]CPFPX PET scans were acquired in 50 healthy human participants (20 females; mean age ± SD 28.0 ± 5.3 years). Mean binding potential (BPND; Logan's reference tissue model with cerebellum as reference region) and volume of distribution (VT) values were calculated in 12 and 15 grey matter brain regions, respectively. [18F]CPFPX BPND was higher in females compared to males in all investigated brain regions (p < 0.025). The largest differences were found in the pallidum and anterior cingulate cortex, where mean BPND values were higher by 29% in females than in males. In females, sleep efficiency correlated positively and sleep latency negatively with BPND in most brain regions. VT values did not differ between sexes. Sleep efficiency correlated positively with VT in most brain regions in female participants. In conclusion, our analysis gives a first indication for potential sex differences in A1AR availability even under well-rested conditions. A1AR availability as measured by [18F]CPFPX BPND is higher in females compared to males. Considering the involvement of adenosine in sleep-wake control, this finding might partially explain the known sex differences in sleep efficiency and sleep latency.

Additional Assessment of Fecal Corticosterone Metabolites Improves Visual Rating in the Evaluation of Stress Responses of Laboratory Rats.

Since animal experiments cannot be completely avoided, the pain, suffering, and distress of laboratory animals must be minimized. To this end, a major prerequisite is reliable assessment of pain and distress. Usually, evaluation of animal welfare is done by visual inspection and score sheets. However, relatively little is known about whether standardized, but subjective, score sheets are able to reliably reflect the status of the animals. The current study aimed to compare visual assessment scores and changes in body weight with concentrations of fecal corticosterone metabolites (FCMs) in a neuroscientific experimental setup. Additionally, effects of refinement procedures were investigated. Eight male adult Sprague-Dawley rats underwent several experimental interventions, including electroencephalograph electrode implantation and subsequent recording, positron emission tomography (PET), and sleep deprivation (SD) by motorized activity wheels. Additional 16 rats were either used as controls without any treatment or to evaluate refinement strategies. Stress responses were determined on a daily basis by means of measuring FCMs, body weight, and evaluation of the animals' welfare by standardized score sheets. Surgery provoked a significant elevation of FCM levels for up to five days. Increases in FCMs due to PET procedures or SD in activity wheels were also highly significant, while visual assessment scores did not indicate elevated stress levels and body weights remained constant. Visual assessment scores correlate with neither changes in body weight nor increases in FCM levels. Habituation procedures to activity wheels used for SD had no impact on corticosterone release. Our results revealed that actual score sheets for visual assessment of animal welfare did not mirror physiological stress responses assessed by FCM measurements. Moreover, small changes in body weight did not correlate with FCM concentration either. In conclusion, as visual assessment is a method allowing immediate interventions on suffering animals to alleviate burden, timely stress assessment in experimental rodents via score sheets should be ideally complemented by validated objective measures (e.g., fecal FCM measured by well-established assays for reliable detection of FCMs). This will complete a comprehensive appraisal of the animals' welfare status in a retrospective manner and refine stressor procedures in the long run.

ADORA2A variation and adenosine A1 receptor availability in the human brain with a focus on anxiety-related brain regions: modulation by ADORA1 variation.

Adenosine, its interacting A1 and A2A receptors, and particularly the variant rs5751876 in the A2A gene ADORA2A have been shown to modulate anxiety, arousal, and sleep. In a pilot positron emission tomography (PET) study in healthy male subjects, we suggested an effect of rs5751876 on in vivo brain A1 receptor (A1AR) availability. As female sex and adenosinergic/dopaminergic interaction partners might have an impact on this rs5751876 effect on A1AR availability, we aimed to (1) further investigate the pilot male-based findings in an independent, newly recruited cohort including women and (2) analyze potential modulation of this rs5751876 effect by additional adenosinergic/dopaminergic gene variation. Healthy volunteers (32/11 males/females) underwent phenotypic characterization including self-reported sleep and A1AR-specific quantitative PET. Rs5751876 and 31 gene variants of adenosine A1, A2A, A2B, and A3 receptors, adenosine deaminase, and dopamine D2 receptor were genotyped. Multivariate analysis revealed an rs5751876 effect on A1AR availability (P = 0.047), post hoc confirmed in 30 of 31 brain regions (false discovery rate (FDR) corrected P values < 0.05), but statistically stronger in anxiety-related regions (e.g., amygdala, hippocampus). Additional effects of ADORA1 rs1874142 were identified; under its influence rs5751876 and rs5751876 × sleep had strengthened effects on A1AR availability (Pboth < 0.02; post hoc FDR-corrected Ps < 0.05 for 29/30 regions, respectively). Our results support the relationship between rs5751876 and A1AR availability. Additional impact of rs1874142, together with rs5751876 and sleep, might be involved in regulating arousal and thus the development of mental disorders like anxiety disorders. The interplay of further detected suggestive ADORA2A × DRD2 interaction, however, necessitates larger future samples more comparable to magnetic resonance imaging (MRI)-based samples.

Impact of acute sleep deprivation on dynamic functional connectivity states.

Sleep deprivation (SD) could amplify the temporal fluctuation of spontaneous brain activities that reflect different arousal levels using a dynamic functional connectivity (dFC) approach. Therefore, we intended to evaluate the test-retest reliability of dFC characteristics during rested wakefulness (RW), and to explore how the properties of these dynamic connectivity states were affected by extended durations of acute sleep loss (28/52 hr). We acquired resting-state fMRI and neuropsychological datasets in two independent studies: (a) twice during RW and once after 28 hr of SD (n = 15) and (b) after 52 hr of SD and after 14 hr of recovery sleep (RS; n = 14). Sliding-window correlations approach was applied to estimate their covariance matrices and corresponding three connectivity states were generated. The test-retest reliability of dFC properties demonstrated mean dwell time and fraction of connectivity states were reliable. After SD, the mean dwell time of a specific state, featured by strong subcortical-cortical anticorrelations, was significantly increased. Conversely, another globally hypoconnected state was significantly decreased. Subjective sleepiness and objective performances were separately positive and negative correlated with the increased and decreased state. Two brain connectivity states and their alterations might be sufficiently sensitive to reflect changes in the dynamics of brain mental activities after sleep loss.

Image-Derived Input Functions for Quantification of A1 Adenosine Receptors Availability in Mice Brains Using PET and [18F]CPFPX.

PURPOSE: In vivo imaging for the A1 adenosine receptors (A1ARs) with positron emission tomography (PET) using 8-cyclopentyl-3-(3-[18F]fluoropropyl)-1-propylxan- thine ([18F]CPFPX) has become an important tool for studying physiological processes quantitatively in mice. However, the measurement of arterial input functions (AIFs) on mice is a method with restricted applicability because of the small total blood volume and the related difficulties in withdrawing blood. Therefore, the aim of this study was to extract an appropriate [18F]CPFPX image-derived input function (IDIF) from dynamic PET images of mice. PROCEDURES: In this study, five mice were scanned with [18F]CPFPX for 60 min. Arterial blood samples (n = 7 per animal) were collected from the femoral artery and corrected for metabolites. To generate IDIFs, three different approaches were selected: (A) volume of interest (VOI) placed over the heart (cube, 10 mm); (B) VOI set over abdominal vena cava/aorta region with a cuboid (5 × 5 × 15 mm); and (C) with 1 × 1 × 1 mm voxels on five consecutive slices. A calculated scaling factor (α) was used to correct for partial volume effect; the method of obtaining the total metabolite correction of [18F]CPFPX for IDIFs was developed. Three IDIFs were validated by comparison with AIF. Validation included the following: visual performance; computing area under the curve (AUC) ratios (IDIF/AIF) of whole-blood curves and parent curves; and the mean distribution volume (V T) ratios (IDIF/AIF) of A1ARs calculated by Logan plot and two-tissue compartment model. RESULTS: Compared with the AIF, the IDIF with VOI over heart showed the best performance among the three IDIFs after scaling by 1.77 (α) in terms of visual analysis, AUC ratios (IDIF/AIF; whole-blood AUC ratio, 1.03 ± 0.06; parent curve AUC ratio, 1.01 ± 0.10) and V T ratios (IDIF/AIF; Logan V T ratio, 1.00 ± 0.17; two-tissue compartment model V T ratio, 1.00 ± 0.13) evaluation. The A1ARs distribution of average parametric images was in good accordance to autoradiography of the mouse brain. CONCLUSION: The proposed study provides evidence that IDIF with VOI over heart can replace AIF effectively for quantification of A1ARs using PET and [18F]CPFPX in mice brains.

Phosphocreatine Levels in the Left Thalamus Decline during Wakefulness and Increase after a Nap.

Scientists have hypothesized that the availability of phosphocreatine (PCr) and its ratio to inorganic phosphate (Pi) in cerebral tissue form a substrate of wakefulness. It follows then, according to this hypothesis, that the exhaustion of PCr and the decline in the ratio of PCr to Pi form a substrate of fatigue. We used 31P-magnetic resonance spectroscopy (31P-MRS) to investigate quantitative levels of PCr, the γ-signal of ATP, and Pi in 30 healthy humans (18 female) in the morning, in the afternoon, and while napping (n = 15) versus awake controls (n = 10). Levels of PCr (2.40 mM at 9 A.M.) decreased by 7.0 ± 0.8% (p = 7.1 × 10-6, t = -5.5) in the left thalamus between 9 A.M. and 5 P.M. Inversely, Pi (0.74 mM at 9 A.M.) increased by 17.1 ± 5% (p = 0.005, t = 3.1) and pH levels dropped by 0.14 ± 0.07 (p = 0.002; t = 3.6). Following a 20 min nap after 5 P.M., local PCr, Pi, and pH were restored to morning levels. We did not find respective significant changes in the contralateral thalamus or in other investigated brain regions. Left hemispheric PCr was signficantly lower than right hemispheric PCr only at 5 P.M. in the thalamus and at all conditions in the temporal region. Thus, cerebral daytime-related and sleep-related molecular changes are accessible in vivo Prominent changes were identified in the thalamus. This region is heavily relied on for a series of energy-consuming tasks, such as the relay of sensory information to the cortex. Furthermore, our data confirm that lateralization of brain function is regionally dynamic and includes PCr.SIGNIFICANCE STATEMENT The metabolites phosphocreatine (PCr) and inorganic phosphate (Pi) are assumed to inversely reflect the cellular energy load. This study detected a diurnal decrease of intracellular PCr and a nap-associated reincrease in the left thalamus. Pi behaved inversely. This outcome corroborates the role of the thalamus as a region of high energy consumption in agreement with its function as a gateway that relays and modulates information flow. Conversely to the dynamic lateralization of thalamic PCr, a constantly significant lateralization was observed in other regions. Increasing fatigue over the course of the day may also be a matter of cerebral energy supply. Comparatively fast restoration of that supply may be part of the biological basis for the recreational value of "power napping."

Cognitive impairments by alcohol and sleep deprivation indicate trait characteristics and a potential role for adenosine A1 receptors.

Trait-like differences in cognitive performance after sleep loss put some individuals more at risk than others, the basis of such disparities remaining largely unknown. Similarly, interindividual differences in impairment in response to alcohol intake have been observed. We tested whether performance impairments due to either acute or chronic sleep loss can be predicted by an individual's vulnerability to acute alcohol intake. Also, we used positron emission tomography (PET) to test whether acute alcohol infusion results in an up-regulation of cerebral A1 adenosine receptors (A1ARs), similar to the changes previously observed following sleep deprivation. Sustained attention in the psychomotor vigilance task (PVT) was tested in 49 healthy volunteers (26 ± 5 SD years; 15 females) (i) under baseline conditions: (ii) after ethanol intake, and after either (iii) total sleep deprivation (TSD; 35 hours awake; n = 35) or (iv) partial sleep deprivation (PSD; four nights with 5 hours scheduled sleep; n = 14). Ethanol- versus placebo-induced changes in cerebral A1AR availability were measured in 10 healthy male volunteers (31 ± 9 years) with [18F]8-cyclopentyl-3-(3-fluoropropyl)-1-propylxanthine (CPFPX) PET. Highly significant correlations between the performance impairments induced by ethanol and sleep deprivation were found for various PVT parameters, including mean speed (TSD, r = 0.62; PSD, r = 0.84). A1AR availability increased up to 26% in several brain regions with ethanol infusion. Our studies revealed individual trait characteristics for being either vulnerable or resilient to both alcohol and to sleep deprivation. Both interventions induce gradual increases in cerebral A1AR availability, pointing to a potential common molecular response mechanism.

Effects of Long-Term Caffeine Consumption on the Adenosine A1 Receptor in the Rat Brain: an In Vivo PET Study with [18F]CPFPX.

PURPOSE: Caffeine, a nonselective antagonist of adenosine receptors, is the most popular psychostimulant worldwide. Recently, a protective role of moderate chronic caffeine consumption against neurodegenerative diseases such as Alzheimer's and Parkinson's disease has been discussed. Thus, aim of the present study was an in vivo investigation of effects of long-term caffeine consumption on the adenosine A1 receptor (A1AR) in the rat brain. PROCEDURES: Sixteen adult, male rats underwent five positron emission tomography (PET) scans with the highly selective A1AR radioligand [18F]CPFPX in order to determine A1AR availability. After the first baseline PET scan, the animals were assigned to two groups: Caffeine treatment and control group. The caffeine-treated animals received caffeinated tap water (30 mg/kg bodyweight/day, corresponding to 4-5 cups of coffee per day in humans) for 12 weeks. Subsequently, caffeine was withdrawn and repeated PET measurements were performed on day 1, 2, 4, and 7 of caffeine withdrawal. The control animals were measured according to the same time schedule. RESULTS: At day 1, after 4.4 h of caffeine withdrawal, a significant decrease (- 34.5%, p < 0.001) of whole brain A1AR availability was observed. Unlike all other investigated brain regions in caffeine-treated rats, the hypothalamus and nucleus accumbens showed no significant intraindividual differences between baseline and first withdrawal PET scan. After approximately 27 h of caffeine withdrawal, the region- and group-specific effects disappeared and A1AR availability settled around baseline. CONCLUSIONS: The present study provides evidence that chronic caffeine consumption does not lead to persistent changes in functional availability of cerebral A1ARs which have previously been associated with neuroprotective effects of caffeine. The acute and region-specific decrease in cerebral A1AR availability directly after caffeine withdrawal is most likely caused by residual amounts of caffeine metabolites disguising an unchanged A1AR expression at this early time-point.

Recovery sleep after extended wakefulness restores elevated A1 adenosine receptor availability in the human brain.

Adenosine and functional A1 adenosine receptor (A1AR) availability are supposed to mediate sleep-wake regulation and cognitive performance. We hypothesized that cerebral A1AR availability after an extended wake period decreases to a well-rested state after recovery sleep. [18F]CPFPX positron emission tomography was used to quantify A1AR availability in 15 healthy male adults after 52 h of sleep deprivation and following 14 h of recovery sleep. Data were additionally compared with A1AR values after 8 h of baseline sleep from an earlier dataset. Polysomnography, cognitive performance, and sleepiness were monitored. Recovery from sleep deprivation was associated with a decrease in A1AR availability in several brain regions, ranging from 11% (insula) to 14% (striatum). A1AR availabilities after recovery did not differ from baseline sleep in the control group. The degree of performance impairment, sleepiness, and homeostatic sleep-pressure response to sleep deprivation correlated negatively with the decrease in A1AR availability. Sleep deprivation resulted in a higher A1AR availability in the human brain. The increase that was observed after 52 h of wakefulness was restored to control levels during a 14-h recovery sleep episode. Individuals with a large increase in A1AR availability were more resilient to sleep-loss effects than those with a subtle increase. This pattern implies that differences in endogenous adenosine and A1AR availability might be causal for individual responses to sleep loss.

Circadian variation of metabotropic glutamate receptor 5 availability in the rat brain.

The metabotrophic subtype 5 glutamate receptor (mGluR5) plays a critical role in synaptic plasticity besides its involvement in numerous neurological disorders, such as depression. As mGluR5 availability in humans is altered in sleep deprivation, we hypothesized that mGluR5 availability underlies a circadian variation. To investigate whether mGluR5 underlies potential circadian changes we measured its density in a randomized fashion at six different daytimes in 11 adult Sprague-Dawley rats. mGluR5 density was quantified by positron emission tomography (PET) using the radioactive ligand [11 C]ABP688. [11 C]ABP688 uptake was quantified in nine regions of interest with a reference tissue model. Significant differences in the binding potential (BPND ) and therefore mGluR5 availability between the different circadian times were found in cortex, cingulate cortex, amygdala, caudate putamen and nucleus accumbens. Further post-hoc statistical analysis (Tukey-Kramer test) of the different time-points revealed significant changes in BPND between 07:00 hours (start of light-on phase) and 15:00 hours (last time-point of the light-on phase) in the caudate putamen. This study shows that mGluR5 availability is increased during the light-on, or sleep phase, of rodents by approximately 10%. Given that altered mGluR5 densities play a role in psychiatric disorders, further investigation is warranted to evaluate their circadian involvement in mood changes in humans.

Chronic sleep restriction induces long-lasting changes in adenosine and noradrenaline receptor density in the rat brain.

Although chronic sleep restriction frequently produces long-lasting behavioural and physiological impairments in humans, the underlying neural mechanisms are unknown. Here we used a rat model of chronic sleep restriction to investigate the role of brain adenosine and noradrenaline systems, known to regulate sleep and wakefulness, respectively. The density of adenosine A1 and A2a receptors and ß-adrenergic receptors before, during and following 5 days of sleep restriction was assessed with autoradiography. Rats (n = 48) were sleep-deprived for 18 h day(-1) for 5 consecutive days (SR1-SR5), followed by 3 unrestricted recovery sleep days (R1-R3). Brains were collected at the beginning of the light period, which was immediately after the end of sleep deprivation on sleep restriction days. Chronic sleep restriction increased adenosine A1 receptor density significantly in nine of the 13 brain areas analysed with elevations also observed on R3 (+18 to +32%). In contrast, chronic sleep restriction reduced adenosine A2a receptor density significantly in one of the three brain areas analysed (olfactory tubercle which declined 26-31% from SR1 to R1). A decrease in ß-adrenergic receptors density was seen in substantia innominata and ventral pallidum which remained reduced on R3, but no changes were found in the anterior cingulate cortex. These data suggest that chronic sleep restriction can induce long-term changes in the brain adenosine and noradrenaline receptors, which may underlie the long-lasting neurocognitive impairments observed in chronic sleep restriction.

Association of adenosine receptor gene polymorphisms and in vivo adenosine A1 receptor binding in the human brain.

Adenosine A1 receptors (A1ARs) and the interacting adenosine A2A receptors are implicated in neurological and psychiatric disorders. Variants within the corresponding genes ADORA1 and ADORA2A were shown associated with pathophysiologic alterations, particularly increased anxiety. It is unknown so far, if these variants might modulate the A1AR distribution and availability in different brain regions. In this pilot study, the influence of ADORA1 and ADORA2A variants on in vivo A1AR binding was assessed with the A1AR-selective positron emission tomography (PET) radioligand [(18)F]CPFPX in brains of healthy humans. Twenty-eight normal control subjects underwent PET procedures to calculate the binding potential BPND of [(18)F]CPFPX in cerebral regions and to assess ADORA1 and ADORA2A single nucleotide polymorphism (SNP) effects on regional BPND data. Our results revealed SNPs of both genes associated with [(18)F]CPFPX binding to the A1AR. The strongest effects that withstood even Bonferroni correction of multiple SNP testing were found in non-smoking subjects (N=22) for ADORA2A SNPs rs2236624 and rs5751876 (corr. Pall<0.05). SNP alleles previously identified at risk for increased anxiety like the rs5751876 T-allele corresponded to consistently higher A1AR availability in all brain regions. Our data indicate for the first time that variation of A1AR availability was associated with ADORA SNPs. The finding of increased A1AR availability in regions of the fear network, particularly in ADORA2A risk allele carriers, strongly warrants evaluation and replication in further studies including individuals with increased anxiety.

Reproducibility of non-invasive a1 adenosine receptor quantification in the rat brain using [(18)F]CPFPX and positron emission tomography.

PURPOSE: The A1AR antagonist 8-cyclopentyl-3-(3-fluoropropyl)-1-propylxanthine ([(18)F]CPFPX) has recently been shown to be a suitable radiotracer for quantitative in vivo imaging of the A1 adenosine receptor (A1AR) in rats. The present study evaluates the reproducibility of non-invasive longitudinal A1AR studies with [(18)F]CPFPX and a dedicated small animal positron emission tomography (PET) scanner. PROCEDURES: Twelve male Sprague Dawley rats underwent four repeated dynamic PET scans with a bolus injection of [(18)F]CPFPX. A1AR availability was determined by different non-invasive approaches including simplified and multilinear reference tissue (olfactory bulb)-based models and graphical methods. The outcome parameter binding potential (BP) was evaluated in terms of variability and reproducibility. RESULTS: Repeated estimations of [(18)F]CPFPX BP ND gave reliable results with acceptable variability (mean 12 %) and reproducibility (intraclass correlation coefficients raging from 0.57 to 0.68) in cortical and subcortical regions of the rat brain. With regard to kinetic models, test-retest stability of the simplified reference-tissue model (SRTM) was superior to multilinear and graphical approaches. CONCLUSIONS: Non-invasive quantification of A1AR density in the rat brain is reproducible and reliable with [(18)F]CPFPX PET and allows longitudinal designs of in vivo imaging studies in rodents.

[¹8F]Altanserin and small animal PET: impact of multidrug efflux transporters on ligand brain uptake and subsequent quantification of 5-HT2A receptor densities in the rat brain.

INTRODUCTION: The selective 5-hydroxytryptamine type 2a receptor (5-HT(2A)R) radiotracer [(18)F]altanserin is a promising ligand for in vivo brain imaging in rodents. However, [(18)F]altanserin is a substrate of P-glycoprotein (P-gp) in rats. Its applicability might therefore be constrained by both a differential expression of P-gp under pathological conditions, e.g. epilepsy, and its relatively low cerebral uptake. The aim of the present study was therefore twofold: (i) to investigate whether inhibition of multidrug transporters (MDT) is suitable to enhance the cerebral uptake of [(18)F]altanserin in vivo and (ii) to test different pharmacokinetic, particularly reference tissue-based models for exact quantification of 5-HT(2A)R densities in the rat brain. METHODS: Eighteen Sprague-Dawley rats, either treated with the MDT inhibitor cyclosporine A (CsA, 50 mg/kg, n=8) or vehicle (n=10) underwent 180-min PET scans with arterial blood sampling. Kinetic analyses of tissue time-activity curves (TACs) were performed to validate invasive and non-invasive pharmacokinetic models. RESULTS: CsA application lead to a two- to threefold increase of [(18)F]altanserin uptake in different brain regions and showed a trend toward higher binding potentials (BP(ND)) of the radioligand. CONCLUSIONS: MDT inhibition led to an increased cerebral uptake of [(18)F]altanserin but did not improve the reliability of BP(ND) as a non-invasive estimate of 5-HT(2A)R. This finding is most probable caused by the heterogeneous distribution of P-gp in the rat brain and its incomplete blockade in the reference region (cerebellum). Differential MDT expressions in experimental animal models or pathological conditions are therefore likely to influence the applicability of imaging protocols and have to be carefully evaluated.

In vivo kinetic and steady-state quantification of 18F-CPFPX binding to rat cerebral A1 adenosine receptors: validation by displacement and autoradiographic experiments.

UNLABELLED: In vivo imaging of the A1 adenosine receptor (A1AR) using (18)F-8-cyclopentyl-3-(3-fluoropropyl)-1-propylxanthine ((18)F-CPFPX) and PET has become an important tool for studying physiologic and pathologic states of the human brain. However, dedicated experimental settings for small-animal studies are still lacking. The aim of the present study was therefore to develop and evaluate suitable pharmacokinetic models for the quantification of the cerebral A1AR in high-resolution PET. METHODS: On a dedicated animal PET scanner, 15 rats underwent (18)F-CPFPX PET scans of 120-min duration. In all animals, arterial blood samples were drawn and corrected for metabolites. The radioligand was injected either as a bolus or as a bolus plus constant infusion. For the definition of unspecific binding, the A1AR selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) was applied. After PET, the brains of 9 animals were dissected and in vitro saturation binding was performed using high-resolution (3)H-DPCPX autoradiography. RESULTS: The kinetics of (18)F-CPFPX were well described by either compartmental or noncompartmental models based on arterial input function. The resulting distribution volume ratio correlated with a low bias toward identity with the binding potential derived from a reference region (olfactory bulb) approach. Furthermore, PET quantification correlated significantly with autoradiographic in vitro data. Blockade of the A1AR with DPCPX identified specific binding of about 45% in the reference region olfactory bulb. CONCLUSION: The present study provides evidence that (18)F-CPFPX PET based on a reference tissue approach can be performed quantitatively in rodents in selected applications. Specific binding in the reference region needs careful consideration for quantitative investigations.