Transgenic inhibition of neuronal calcineurin activity in the forebrain facilitates fear conditioning, but inhibits the extinction of contextual fear memories

It is unclear whether protein phosphatases, which counteract the actions of protein kinases, play a beneficial role in the formation and extinction of previously acquired fear memories. In this study, we investigated the role of the calcium/calmodulin dependent phosphatase 2B, also known as calcineurin (CaN) in the formation of contextual fear memory and extinction of previously acquired contextual fear. We used a temporally regulated transgenic approach, that allowed us to selectively inhibit neuronal CaN activity in the forebrain either during conditioning or only during extinction training leaving the conditioning undisturbed. Reducing CaN activity through the expression of a CaN inhibitor facilitated contextual fear conditioning, while it impaired the extinction of previously formed contextual fear memory. These findings give the first genetic evidence that neuronal CaN plays an opposite role in the formation of contextual fear memories and the extinction of previously formed contextual fear memories. (C) 2007 Elsevier Inc. All rights reserved

Measuring serotonin synthesis: from conventional methods to PET tracers and their (pre)clinical implications

The serotonergic system of the brain is complex, with an extensive innervation pattern covering all brain regions and endowed with at least 15 different receptors (each with their particular distribution patterns), specific reuptake mechanisms and synthetic processes. Many aspects of the functioning of the serotonergic system are still unclear, partially because of the difficulty of measuring physiological processes in the living brain. In this review we give an overview of the conventional methods of measuring serotonin synthesis and methods using positron emission tomography (PET) tracers, more specifically with respect to serotonergic function in affective disorders. Conventional methods are invasive and do not directly measure synthesis rates. Although they may give insight into turnover rates, a more direct measurement may be preferred. PET is a noninvasive technique which can trace metabolic processes, like serotonin synthesis. Tracers developed for this purpose are α-[11C]methyltryptophan ([11C]AMT) and 5-hydroxy-L-[β-11C]tryptophan ([11C]5-HTP). Both tracers have advantages and disadvantages. [11C]AMT can enter the kynurenine pathway under inflammatory conditions (and thus provide a false signal), but this tracer has been used in many studies leading to novel insights regarding antidepressant action. [11C]5-HTP is difficult to produce, but trapping of this compound may better represent serotonin synthesis. AMT and 5-HTP kinetics are differently affected by tryptophan depletion and changes of mood. This may indicate that both tracers are associated with different enzymatic processes. In conclusion, PET with radiolabelled substrates for the serotonergic pathway is the only direct way to detect changes of serotonin synthesis in the living brain

Similar serotonin-2A receptor binding in rats with different coping styles or levels of aggression

Individual differences in coping style emerge as a function of underlying variability in the activation of a mesocorticolimbic brain circuitry. Particularly serotonin seems to play an important role. For this reason, we assessed serotonin-2A receptor (5-HT2AR) binding in the brain of rats with different coping styles. We compared proactive and reactive males of two rat strains, Wild-type Groningen (WTG) and Roman high-and low avoidance (RHA, RLA). 5-HT2AR binding in (pre)frontal cortex (FC) and hippocampus was investigated using a radiolabeled antagonist ([H-3]MDL-100907) and agonist ([H-3]Cimbi-36) in binding assays. No differences in 5-HT2AR binding were observed in male animals with different coping styles. [H-3] MDL-100907 displayed a higher specific-to-nonspecific binding ratio than [H-3] Cimbi-36. Our findings suggest that in these particular rat strains, 5-HT2AR binding is not an important molecular marker for coping style. Because neither an antagonist nor an agonist tracer showed any binding differences, it is unlikely that the affinity state of the 5-HT2AR is co-varying with levels of aggression or active avoidance in WTG, RHA and RLA. (C) 2015 Wiley Periodicals, Inc

Acute social defeat does not alter cerebral 5-HT2A receptor binding in male Wistar rats

It has been hypothesized that effects of uncontrollable stress on serotonin receptor expression contribute to the etiology of stress-related disorders like depression. While the serotonin-2A receptors (5-HT2AR) are thought to be important in this context, only few studies examined effects of stress on this receptor subtype. In this study, we therefore assessed acute and long-term changes in 5HT(2A)R binding after social defeat stress in rats. Male Wistar rats were subjected to social defeat by placing them in the home cage of an aggressive, dominant Long Evans rat. Acute social defeat suppressed growth, but did not affect anxiety-like behavior in an open field test. A positron emission tomography scan with the 5-HT2AR tracer [C-11]MDL 100907 1 day and 3 weeks after defeat did not show significant changes in receptor binding. To verify these results, [H-3]MDL 100907 binding assays were performed in homogenates of prefrontal cortex and hippocampus, which also did not indicate any changes in B-max or K-d. These findings do not support the hypothesis that changes in 5-HT2AR function are a vital mechanism through which uncontrollable stress contributes to stress-related pathologies such as depression. It remains to be determined whether effects of stress on 5HT(2A)R binding depend on the nature of the stressor or on the characteristics of the rat strain. (C) 2014 Wiley Periodicals, Inc

[(11)C]5-HTP and microPET are not suitable for pharmacodynamic studies in the rodent brain

The PET tracer [(11)C]5-hydroxytryptophan ([(11)C]5-HTP), which is converted to [(11)C]5-hydroxytryptamine ([(11)C]5-HT) by aromatic amino acid decarboxylase (AADC), is thought to measure 5-HT synthesis rates. But can we measure these synthesis rates by kinetic modeling of [(11)C]5-HTP in rat? Male rats were scanned with [(11)C]5-HTP (60 minutes) after different treatments. Scans included arterial blood sampling and metabolite analysis. 5-HT synthesis rates were calculated by a two-tissue compartment model (2TCM) with irreversible tracer trapping or Patlak analysis. Carbidopa (inhibitor peripheral AADC) dose-dependently increased [(11)C]5-HTP brain uptake, but did not influence 2TCM parameters. Therefore, 10 mg/kg carbidopa was applied in all subsequent study groups. These groups included treatment with NSD 1015 (general AADC inhibitor) or p-chlorophenylalanine (PCPA, inhibitor of tryptophan hydroxylase, TPH). In addition, the effect of a low-tryptophan (Trp) diet was investigated. NSD 1015 or Trp depletion did not affect any model parameters, but PCPA reduced [(11)C]5-HTP uptake, and the k(3). This was unexpected as NSD 1015 directly inhibits the enzyme converting [(11)C]5-HTP to [(11)C]5-HT, suggesting that trapping of radioactivity does not distinguish between parent tracer and its metabolites. As different results have been acquired in monkeys and humans, [(11)C]5-HTP-PET may be suitable for measuring 5-HT synthesis in primates, but not in rodents

Dose-dependent sigma-1 receptor occupancy by donepezil in rat brain can be assessed with (11)C-SA4503 and microPET

RATIONALE: Sigma-1 receptor agonists are under investigation as potential disease-modifying agents for several CNS disorders. Donepezil, an acetylcholinesterase inhibitor used for the symptomatic treatment of Alzheimer's disease, is also a high-affinity sigma-1 agonist. OBJECTIVES: The objectives of the present study were to investigate if the sigma-1 agonist tracer (11)C-SA4503 and microPET can be used to determine sigma-1 receptor occupancy (RO) of donepezil in the rat brain; to establish RO of donepezil at doses commonly used in rodent behavioural studies; and to determine the effective plasma concentration of donepezil required for 50 % of max-min occupancy (EC50). METHODS: Male Wistar rats were pre-treated with donepezil (0.1 to 10 mg/kg) for about 1 h before microPET scans using (11)C-SA4503. The total distribution volume (V T) of the tracer was determined by Logan graphical analysis using time activity curves from arterial plasma and regions of interest drawn around the entire brain and individual brain regions. RO by donepezil was calculated from a modified Lassen plot, and ED50 was estimated from the sigmoidal dose-response curves obtained when the RO was plotted against log donepezil dose. RESULTS: A dose-dependent reduction was observed for V T in the whole brain as well as individual brain regions. RO increased dose-dependently and was 93 % at 10 mg/kg. ED50 was 1.29 mg/kg. CONCLUSIONS: Donepezil, in the common dose range, was found to dose-dependently occupy a significant fraction of the sigma-1 receptor population. The data indicate that it is possible to determine sigma-1 RO by an agonist drug in rat brain, using (11)C-SA4503 and microPET

Serotonin-2C antagonism augments the effect of citalopram on serotonin and dopamine levels in the ventral tegmental area and nucleus accumbens

Many patients with major depression do not respond to selective serotonin reuptake inhibitors (SSRIs). Lack of response could be due to inhibition of dopamine (DA) release by serotonin (5-HT) through 5-HT2c receptors. Combining an SSRI with a 5-HT2c antagonist may result in improved efficacy by causing simultaneous increases of 5-HT and DA. In order to test this augmentation strategy, male Wistar rats were treated (s.c.) with an acute dose of the SSRI citalopram (Cit, 5 mg/ kg), the 5-HT2c antagonist SB 242084 (SB, 2 mg/ kg), or Cit + SB, and the effect on 5-HT and DA release in the nucleus accumbens (NAcc) was assessed by microdialysis. In a separate experiment, animals were treated with vehicle, Cit (20 mg/kg/ d), SB (2 mg/kg/d) or Cit + SB for a period of 2 days (s.c.), and the impact on the release of 5-HT and DA in the ventral tegmental area (VIA) and NAcc was studied. On the day of microdialysis, 5-HT2c receptor sensitivity was assessed with an SB challenge. Acutely administered Cit + SB increased 5-HT release in the NAcc more than Cit alone. SB alone increased DA release in the NAcc (not in the VTA), but when administered together with Cit, this effect was abolished. A 2-day treatment with Cit or Cit + SB increased 5-HT release in both VTA and NAcc. Combining Cit with SB augmented the effect of Cit in the VTA. DA release in VTA and NAcc was only significantly increased after 2-days of treatment with Cit + SB. In conclusion, Cit + SB had synergistic effects on 5-HT and DA release after 2-days of treatment, probably related to a decreased tonic inhibition of DA release via 5-HT2c receptors. Regional differences occur and future studies should elucidate if this augmentation strategy is beneficial at the behavioral level. (C) 2014 Elsevier Ltd. All rights reserved

Small-Animal PET with a sigma-Ligand, C-11-SA4503, Detects Spontaneous Pituitary Tumors in Aged Rats

<p>Pituitary tumors are often detected only after death or at late stages of the disease when they are macroadenomas with a low surgical cure rate. Spontaneous pituitary tumors occur in rats over 1 y of age. In an ongoing study of changes in sigma-1 agonist binding related to aging, several of our rats developed such tumors. The aim of the current study was to assess the kinetics of C-11-SA4503 C-11-labeled 1-[2-(3,4-dimethoxyphenthyl)]-4-(3-phenylpropyl)-piperazine dihydrochloride) in tumor and brain and to evaluate the utility of this tracer in the detection of pituitary tumors. Methods: Small-animal PET scans of the brain region of male Wistar Hannover rats (age, 18-32 mo) were acquired using the sigma-1 agonist tracer C-11-SA4503. The time-dependent uptake of C-11 in the entire brain, tumor or normal pituitary, and thyroid was measured. A 2-tissue-compartment model was fitted to the PET data, using metabolite-corrected plasma radioactivity as the input function. Results: Pituitary tumors showed up as bright hot spots in the scans. The total distribution volume (V-T) of the tracer was significantly higher in the tumor than in the normal pituitary. Surprisingly, a higher V-T was also seen in the brain and thyroid tissue of animals with pituitary tumors than in healthy rats. The increase in V-T in the brain and thyroid was not related to a change in nondisplaceable binding potential (BPND) but rather to an increase in the partition coefficient (K-1/k(2)) of C-11-SA4503. The increase in V-T in the tumor on the other hand was accompanied by a significant increase in BPND. Western blotting analysis indicated that pituitary tumors overexpressed sigma-1 receptors. Conclusion: The overexpression of sigma-1 receptors in spontaneous pituitary tumors is detected as an increase in uptake and BPND of C-11-SA4503. Therefore, this tracer may have promise for the detection of pituitary adenomas, using PET.</p>