Detecting a dexmedetomidine‐evoked reduction of noradrenaline release in the human brain with the alpha2C‐adrenoceptor PET ligand [11C]ORM‐13070

PET imaging can for some neurotransmitters be used to measure synaptic neurotransmitter concentrations. The objective of this study was to test whether the receptor binding of the α_2C‐AR antagonist PET tracer [¹¹C]ORM‐13070 would increase in response to reductions in synaptic noradrenaline, evoked by dexmedetomidine as a sympatholytic drug challenge. Six subjects underwent a control PET scan and two dexmedetomidine PET scans. Dexmedetomidine was infused with target plasma concentrations of 0.6 and 0.2 ng/ml. Tracer binding was measured by voxel‐based analysis of bound per free (B/F) images. ROI‐based analysis was performed in the dorsal striatum and in the thalamus. Vital signs and drug concentrations in plasma were measured and the sedative effect was estimated with the visual analog scale. In the voxel‐based analysis, dexmedetomidine administration was associated with a tendency to increased B/F tracer in the right thalamus (mean, +17%, P = 0.14, and +19%, P = 0.05, with the low and high dose, respectively). Tracer binding in the dorsal striatum was unaffected by dexmedetomidine. A cluster with significantly increased B/F tracer (+42%, P = 0.01) was seen in the right superior temporal gyrus with low‐dose dexmedetomidine, but not after the high dose. Brain uptake of [¹¹C]ORM‐13070 has previously been shown to be reduced in conditions of increased synaptic noradrenaline concentrations. In this study, tracer binding in the thalamus tended to increase in accordance with reduced activity of noradrenergic projections from the locus coeruleus, but statistical significance was not reached. Synapse 70:57–65, 2016. © 2015 Wiley Periodicals, Inc.     

Regional distribution and cellular localization of β2‐adrenoceptors in the adult zebrafish brain (Danio rerio)

The β₂‐adrenergic receptors (ARs) are G‐protein‐coupled receptors that mediate the physiological responses to adrenaline and noradrenaline. The present study aimed to determine the regional distribution of β₂‐ARs in the adult zebrafish (Danio rerio) brain by means of in vitro autoradiographic and immunohistochemical methods. The immunohistochemical localization of β₂‐ARs, in agreement with the quantitative β‐adrenoceptor autoradiography, showed a wide distribution of β₂‐ARs in the adult zebrafish brain. The cerebellum and the dorsal zone of periventricular hypothalamus exhibited the highest density of [³H]CGP‐12177 binding sites and β₂‐AR immunoreactivity. Neuronal cells strongly stained for β₂‐ARs were found in the periventricular ventral telencephalic area, magnocellular and parvocellular superficial pretectal nuclei (PSm, PSp), occulomotor nucleus (NIII), locus coeruleus (LC), medial octavolateral nucleus (MON), magnocellular octaval nucleus (MaON) reticular formation (SRF, IMRF, IRF), and ganglionic cell layer of cerebellum. Interestingly, in most cases (NIII, LC, MON, MaON, SRF, IMRF, ganglionic cerebellar layer) β₂‐ARs were colocalized with α_2A‐ARs in the same neuron, suggesting their interaction for mediating the physiological functions of nor/adrenaline. Moderate to low labeling of β₂‐ARs was found in neurons in dorsal telencephalic area, optic tectum (TeO), torus semicircularis (TS), and periventricular gray zone of optic tectum (PGZ). In addition to neuronal, glial expression of β₂‐ARs was found in astrocytic fibers located in the central gray and dorsal rhombencephalic midline, in close relation to the ventricle. The autoradiographic and immunohistochemical distribution pattern of β₂‐ARs in the adult zebrafish brain further support the conserved profile of adrenergic/noradrenergic system through vertebrate brain evolution. J. Comp. Neurol. 518:1418–1441, 2010. © 2009 Wiley‐Liss, Inc.     

Coexpression of α2A‐adrenergic and δ‐opioid receptors in substance P‐containing terminals in rat dorsal horn

Agonists acting at α₂‐adrenergic and opioid receptors (α₂ARs and ORs, respectively) inhibit pain transmission in the spinal cord. When coadministered, agonists activating these receptors interact in a synergistic manner. Although the existence of α₂AR/OR synergy has been well characterized, its mechanism remains poorly understood. The formation of heterooligomers has been proposed as a molecular basis for interactions between neuronal G‐protein‐coupled receptors. The relevance of heterooligomer formation to spinal analgesic synergy requires demonstration of the expression of both receptors within the same neuron as well as the localization of both receptors in the same neuronal compartment. We used immunohistochemistry to investigate the spatial relationship between α₂ARs and ORs in the rat spinal cord to determine whether coexpression could be demonstrated between these receptors. We observed extensive colocalization between α_2A‐adrenergic and δ‐opioid receptors (DOP) on substance P (SP)‐immunoreactive (‐ir) varicosities in the superficial dorsal horn of the spinal cord and in peripheral nerve terminals in the skin. α_2AAR‐ and DOP‐ir elements were colocalized in subcellular structures of 0.5 μm or less in diameter in isolated nerve terminals. Furthermore, coincubation of isolated synaptosomes with α₂AR and DOP agonists resulted in a greater‐than‐additive increase in the inhibition of K⁺‐stimulated neuropeptide release. These findings suggest that coexpression of the synergistic receptor pair α_2AAR‐DOP on primary afferent nociceptive fibers may represent an anatomical substrate for analgesic synergy, perhaps as a result of protein–protein interactions such as heterooligomerization. J. Comp. Neurol. 513:385–398, 2009. © 2009 Wiley‐Liss, Inc.     

Synthesis,characterization, and monkey PET studies of [18F]MK‐1312, a PET tracer for quantification of mGluR1 receptor occupancy by MK‐5435

Two moderately lipophilic, high affinity ligands for metabotropic glutamate receptor subtype 1 (mGluR1) were radiolabeled with a positron‐emitting radioisotope and evaluated in rhesus monkey as potential PET tracers. Both ligands were radiolabeled with fluorine‐18 via nucleophilic displacement of the corresponding 2‐chloropyridine precursor with [¹⁸F]potassium fluoride. [¹⁸F] MK‐1312 was found to have a suitable signal for quantification of mGluR1 receptors in nonhuman primates and was more thoroughly characterized. In vitro autoradiographic studies with [¹⁸F] MK‐1312 in rhesus monkey and human brain tissue slices revealed an uptake distribution consistent with the known distribution of mGluR1, with the highest uptake in the cerebellum, moderate uptake in the hippocampus, thalamus, and cortical regions, and lowest uptake in the caudate and putamen. In vitro saturation binding studies in rhesus monkey and human cerebellum homogenates confirmed that [¹⁸F] MK‐1312 binds to a single site with a B_max/K_d ratio of 132 and 98, respectively. PET studies in rhesus monkey with [¹⁸F] MK‐1312 showed high brain uptake and a regional distribution consistent with in vitro autoradiography results. Blockade of [¹⁸F] MK‐1312 uptake with mGluR1 allosteric antagonist MK‐5435 dose‐dependently reduced tracer uptake in all regions of gray matter to a similarly low level of tracer uptake. This revealed a large specific signal useful for determination of mGluR1 receptor occupancy in rhesus monkey. Taken together, these results are promising for clinical PET studies with [¹⁸F] MK‐1312 to determine mGluR1 occupancy of MK‐5435. Synapse 2011. © 2010 Wiley‐Liss, Inc.     

Activation of α1‐adrenoceptors enhances excitatory synaptic transmission via a pre‐ and postsynaptic protein kinase C‐dependent mechanism in the medial prefrontal cortex of rats

The physiological effects of α1‐adrenoceptors (α1‐ARs) have been examined in many brain regions. However, little is known about the mechanism of modulation on synaptic transmission by α1‐ARs in the medial prefrontal cortex (mPFC). The present study investigated how α₁‐AR activation regulates glutamatergic synaptic transmission in layer V/VI pyramidal cells of the rat mPFC. We found that the α₁‐AR agonist phenylephrine (Phe) induced a significant enhancement of the amplitude and frequency of miniature excitatory postsynaptic currents (mEPSCs). The facilitation effect of Phe on the frequency of mEPSCs involved a presynaptic protein kinase C‐dependent pathway. Phe produced a significant enhancement on the amplitude of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor (AMPA‐R)‐ and N‐methyl‐d ‐aspartic acid receptor (NMDA‐R)‐mediated evoked excitatory postsynaptic currents (eEPSCs). Phe enhanced inward currents evoked by puff application of glutamate or NMDA. The Phe‐induced facilitation of AMPA‐R‐ and NMDA‐R‐mediated eEPSCs required, in part, postsynaptic G_q, phospholipase C and PKC. These findings suggest that α₁‐AR activation facilitates excitatory synaptic transmission in mPFC pyramidal cells via both pre‐ and post‐synaptic PKC‐dependent mechanisms.     

Noradrenaline‐mediated facilitation of inhibitory synaptic transmission in the dorsal horn of the rat spinal cord involves interlaminar communications

In the dorsal horn of the spinal cord (DH), noradrenaline (NA) is released by axons originating from the locus coeruleus and induces spinal analgesia, the mechanisms of which are poorly understood. Here, the effects of NA on synaptic transmission in the deep laminae (III–V) of the DH were characterized. It was shown that exogenously applied, as well as endogenously released, NA facilitated inhibitory [γ‐aminobutyric acid (GABA)ergic and glycinergic] synaptic transmission in laminae III–IV of the DH by activating α₁‐, α₂‐ and β‐adrenoceptors (ARs). In contrast, NA had no effect on excitatory (glutamatergic) synaptic transmission. Physical interruption of communications between deep and more superficial laminae (by a mechanical transection between laminae IV and V) totally blocked the effects of α₂‐AR agonists and strongly reduced the effects of α₁‐AR agonists on inhibitory synaptic transmission in laminae III–IV without directly impairing synaptic release of GABA or glycine from neurons. Short‐term pretreatment of intact spinal cord slices with the glial cell metabolism inhibitor fluorocitrate or pharmacological blockade of ionotropic glutamate and ATP receptors mimicked the consequences of a mechanical transection between laminae IV and V. Taken together, the current results indicate that the facilitation of inhibitory synaptic transmission in laminae III–IV of the DH by NA requires functional interlaminar connections between deep and more superficial laminae, and might strongly depend on glia to neuron interactions. These interlaminar connections and glia to neuron interactions could represent interesting targets for analgesic strategies.     

Evaluation of (‐)‐[18F]Flubatine‐specific binding: Implications for reference region approaches

We aimed to characterize changes in binding of (‐)‐[¹⁸F]Flubatine to α₄β₂*‐nicotinic acetylcholine receptors (α₄β₂*‐nAChRs) during a tobacco cigarette smoking challenge. Displacement of (‐)‐[¹⁸F]Flubatine throughout the brain was quantified as change in (‐)‐[¹⁸F]Flubatine distribution volume (V_T), with particular emphasis on regions with low V_T. Three tobacco smokers were imaged with positron emission tomography (PET) during a 210 min bolus‐plus‐constant infusion of (‐)‐[¹⁸F]Flubatine. A tobacco cigarette was smoked in the PET scanner ～125 min after the start of (‐)‐[¹⁸F]Flubatine injection. Equilibrium analysis was used to estimate V_T at baseline (90‐120 min) and after cigarette challenge (180‐210 min), at the time of greatest receptor occupancy by nicotine. Smoking reduced V_T by 21 ± 9% (average ±SD) in corpus callosum, 17 ± 9% in frontal cortex, 36 ± 11% in cerebellum, and 22 ± 10% in putamen. The finding of displaceable (‐)‐[¹⁸F]Flubatine binding throughout the brain is an important consideration for reference region‐based quantification approaches with this tracer. We observed displacement of (‐)‐[¹⁸F]Flubatine binding to α₄β₂*‐nicotinic acetylcholine receptors in corpus callosum by a tobacco cigarette challenge. We conclude that reference region approaches utilizing corpus callosum should first perform careful characterization of displaceable (‐)‐[¹⁸F]Flubatine binding and nondisplaceable kinetics in this putative reference region.     

Brain PET measurement of PDE10A occupancy by TAK‐063, a new PDE10A inhibitor,using [11C]T‐773 in nonhuman primates

Because phosphodiesterase 10A (PDE10A) degrades both cyclic adenosine monophosphate and cyclic guanosine monophosphate and is distributed mainly in the striatum, PDE10A inhibitors have been considered to potentially be useful therapeutic agents for psychiatric and neurodegenerative diseases such as schizophrenia and Huntington's disease. We measured striatal PDE10A occupancy by TAK‐063, a newly developed compound with high affinity and selectivity for PDE10A, using PET with [¹¹C]T‐773 in nonhuman primates. Two 123‐min dynamic PET measurements were performed on three female rhesus monkeys, once at baseline and again after intravenous administration of different doses of TAK‐063 (0.2–1.6 mg/kg). Total distribution volume (V_T) was calculated with a two‐tissue compartment model using metabolite‐corrected plasma input. Although the in vitro autoradiography did not show high specific binding to [¹¹C]T‐773 in the cerebellum, V_T in the cerebellum decreased after TAK‐063 treatment. The specific binding to PDE10A (V_S) was calculated as the difference of the V_T between the target regions and the cerebellum. PDE10A occupancy was calculated as the percent change of V_S. The average PDE10A occupancy of the caudate nucleus and putamen was 35.2% at 0.2 mg/kg and 83.2% at 1.6 mg/kg. In conclusion, this nonhuman primate PET study demonstrated that [¹¹C]T‐773 is useful to estimate the PDE10A occupancy by TAK‐063 in the striatum although there is in vivo interaction of the uptake between [¹¹C]T‐773 and TAK‐063 in the cerebellum. These results warrant further clinical occupancy study for TAK‐063. Synapse 70:253–263, 2016 . © 2016 Wiley Periodicals, Inc.     

Comparative evaluation of two glycine transporter 1 radiotracers [11C]GSK931145 and [18F]MK‐6577 in baboons

Glycine transporter type‐1 (GlyT1) has been proposed as a target for drug development for schizophrenia. PET imaging with a GlyT1 specific radiotracer will allow for the measurement of target occupancy of GlyT1 inhibitors, and for in vivo investigation of GlyT1 alterations in schizophrenia. We conducted a comparative evaluation of two GlyT1 radiotracers, [¹¹C]GSK931145, and [¹⁸F]MK‐6577, in baboons. Two baboons were imaged with [¹¹C]GSK931145 and [¹⁸F]MK‐6577. Blocking studies with GSK931145 (0.3 or 0.2 mg/kg) were conducted to determine the level of tracer specific binding. [¹¹C]GSK931145 and [¹⁸F]MK‐6577 were synthesized in good yield and high specific activity. Moderately fast metabolism was observed for both tracers, with ～30% of parent at 30 min post‐injection. In the brain, both radiotracers showed good uptake and distribution profiles consistent with regional GlyT1 densities. [¹⁸F]MK‐6577 displayed higher uptake and faster kinetics than [¹¹C]GSK931145. Time activity curves were well described by the two‐tissue compartment model. Regional volume of distribution (V_T) values were higher for [¹⁸F]MK‐6577 than [¹¹C]GSK931145. Pretreatment with GSK931145 reduced tracer uptake to a homogeneous level throughout the brain, indicating in vivo binding specificity and lack of a reference region for both radiotracers. Linear regression analysis of V_T estimates between tracers indicated higher specific binding for [¹⁸F]MK‐6577 than [¹¹C]GSK931145, consistent with higher regional binding potential (BP_ND) values of [¹⁸F]MK‐6577 calculated using V_T from the baseline scans and non‐displaceable distribution volume (V_ND) derived from blocking studies. [¹⁸F]MK‐6577 appears to be a superior radiotracer with higher brain uptake, faster kinetics, and higher specific binding signals than [¹¹C]GSK931145. Synapse 70:112–120, 2016. © 2016 Wiley Periodicals, Inc.     

Inverse agonist histamine H3 receptor PET tracers labelled with carbon‐11 or fluorine‐18

Two histamine H3 receptor (H3R) inverse agonist PET tracers have been synthesized and characterized in preclinical studies. Each tracer has high affinity for the histamine H3 receptor, has suitable lipophilicity, and neither is a substrate for the P‐glycoprotein efflux pump. A common phenolic precursor was used to synthesize each tracer with high specific activity and radiochemical purity by an alkylation reaction using either [¹¹C]MeI or [¹⁸F]FCD₂Br. Autoradiographic studies in rhesus monkey and human brain slices showed that each tracer had a widespread distribution with high binding densities in frontal cortex, globus pallidus and striatum, and lower uptake in cerebellum. The specificity of this expression pattern was demonstrated by the blockade of the autoradiographic signal by either the H3R agonist R‐α‐methylhistamine or a histamine H3R inverse agonist. In vivo PET imaging studies in rhesus monkey showed rapid uptake of each tracer into the brain with the same distribution seen in the autoradiographic studies. Each tracer could be blocked by pretreatment with a histamine H3R inverse agonist giving a good specific signal. Comparison of the in vitro metabolism of each compound showed slower metabolism in human liver microsomes than in rhesus monkey liver microsomes, with each compound having a similar clearance rate in humans. The in vivo metabolism of 1b in rhesus monkey showed that at 60 min, ～35% of the circulating counts were due to the parent. These tracers are very promising candidates as clinical PET tracers to both study the histamine H3R system and measure receptor occupancy of H3R therapeutic compounds. Synapse 63:1122–1132, 2009. © 2009 Wiley‐Liss, Inc.     

Effects of the conformationally restricted GABA analogues,Cis‐and Trans‐4‐aminocrotonic acid,on GABA neurotransmission in primary neuronal cultures

The effects of the GABA analogues, cis‐ and trans‐4‐aminocrotonic acid (ACA) on GABA_A receptor function and GABA uptake, together with the presence of ρ‐1 subunit mRNA and putative GABA_C receptors, were studied in primary cultures of neocortical neurons and cerebellar granule cells. Both isomers induced a Cl^‐ influx, which was inhibited by bicuculline, t‐butylbicyclophosphorothionate (TBPS), picrotoxinin (PTX), and γ‐hexachlorocyclohexane (γ‐HCH or lindane). [³H]‐flunitrazepam binding was also increased by both isomers and this increase was inhibited by bicuculline. In neocortical neurons, the trans‐isomer completely inhibited the [³H]GABA uptake, whereas the cis‐isomer produced only a 25% inhibition at the highest concentration used. The possible presence of GABA_C receptors was investigated only in neocortical cultures by using RT‐PCR in order to detect the presence of the mRNA encoding the ρ‐1 subunit which assembles to form homooligomeric Cl^‐ channels. The results presented here show that ρ‐1 subunits, and thus GABA_C receptors, may represent a very minor population of GABA receptors in these neuronal preparations. We conclude that both GABA analogues may act as agonists at the GABA_A receptors, although with very different potencies. J. Neurosci. Res. 57:95–105, 1999. © 1999 Wiley‐Liss, Inc.     

Dynamics of β‐adrenergic/cAMP signaling and morphological changes in cultured astrocytes

The morphology of astrocytes, likely regulated by cAMP, determines the structural association between astrocytes and the synapse, consequently modulating synaptic function. β‐Adrenergic receptors (β‐AR), which increase cytosolic cAMP concentration ([cAMP]_i), may affect cell morphology. However, the real‐time dynamics of β‐AR‐mediated cAMP signaling in single live astrocytes and its effect on cell morphology have not been studied. We used the fluorescence resonance energy transfer (FRET)‐based cAMP biosensor Epac1‐camps to study time‐dependent changes in [cAMP]_i; morphological changes in primary rat astrocytes were monitored by real‐time confocal microscopy. Stimulation of β‐AR by adrenaline, noradrenaline, and isoprenaline, a specific agonist of β‐AR, rapidly increased [cAMP]_i (～15 s). The FRET signal response, mediated via β‐AR, was faster than in the presence of forskolin (twofold) and dibutyryl‐cAMP (>35‐fold), which directly activate adenylyl cyclase and Epac1‐camps, respectively, likely due to slow entry of these agents into the cytosol. Oscillations in [cAMP]_i have not been recorded, indicating that cAMP‐dependent processes operate in a slow time domain. Most Epac1‐camps expressing astrocytes revealed a morphological change upon β‐AR activation and attained a stellate morphology within 1 h. The morphological changes exhibited a bell‐shaped dependency on [cAMP]_i. The 5–10% decrease in cell cross‐sectional area and the 30–50% increase in cell perimeter are likely due to withdrawal of the cytoplasm to the perinuclear region and the appearance of protrusions on the surface of astrocytes. Because astrocyte processes ensheath neurons, β‐AR/cAMP‐mediated morphological changes can modify the geometry of the extracellular space, affecting synaptic, neuronal, and astrocyte functions in health and disease. GLIA 2014;62:566–579     

Signaling mechanism underlying α2A‐adrenergic suppression of excitatory synaptic transmission in the medial prefrontal cortex of rats

Stimulation of α_2A‐adrenoceptors (ARs) in the prefrontal cortex (PFC) produces a beneficial effect on cognitive functions such as working memory. A previous study in our laboratory showed that α_2A‐AR stimulation suppresses excitatory synaptic transmission in layer V‐VI pyramidal cells of the rat medial PFC (mPFC). However, the intracellular mechanism underlying the α_2A‐AR suppression remains unclear. In the present study, we recorded evoked excitatory postsynaptic current (eEPSC) in layer V‐VI pyramidal cells of the mPFC, using whole‐cell patch‐clamp recording. We found that the α_2A‐AR agonist guanfacine significantly suppresses eEPSC in mPFC pyramidal cells. The α_2A‐AR inhibition is mediated by the Gi‐cAMP‐PKA‐PP1‐CaMKII‐AMPAR signaling pathway, as such inhibition no longer exists when each step of this pathway is blocked with NF023, Rp‐cAMP, PKI_5–24 or H89, tautomycin, and KN‐62 or KN‐93, respectively.     

Comparing α7 nicotinic acetylcholine receptor binding,amyloid‐β deposition,and mitochondria complex‐I function in living brain: A PET study in aged monkeys

This study was aimed to assess the correlations among α7 nicotinic acetylcholine receptor (α7‐nAChR) binding, amyloid‐β (Aβ) deposition, and mitochondrial complex I (MC‐I) activity in the brain of aged monkeys (Macaca mulatta). Positron emission tomography (PET) measurements with [¹¹C](R)‐MeQAA, [¹¹C]PIB, and [¹⁸F]BCPP‐EF were conducted in monkeys in a conscious condition. [¹¹C](R)‐MeQAA binding was analyzed by a simplified reference tissue model to calculate nondisplaceable binding potential (BP_ND), [¹¹C]PIB uptake was calculated by standard uptake value ratio (SUVR), and [¹⁸F]BCPP‐EF binding was determined by Logan graphical analysis to calculate total distribution volume (V_T) with arterial blood sampling. Higher brain uptake was determined in the thalamus, hippocampus, striatum, and cortical regions for [¹¹C](R)‐MeQAA, while being lower in the cerebellum. Significant age‐related reduction of [¹¹C](R)‐MeQAA binding to α7‐nAChR was determined only in the occipital cortex. The plot of Vt of [¹⁸F]BCPP‐EF against BP_ND of [¹¹C](R)‐MeQAA indicated a significant negative correlation in the hippocampus and cortical regions in aged animals. Plotting of SUVR of [¹¹C]PIB against BP_ND of [¹¹C](R)‐MeQAA showed a positive correlation. The in vivo binding of [¹¹C](R)‐MeQAA could reflect the upregulation of α7‐nAChR induced by neurodegenerative damage determined by Aβ deposition as well as impaired MC‐I activity in living brain. Synapse 69:475–483, 2015. © 2015 Wiley Periodicals, Inc.     

An open‐label positron emission tomography study to evaluate serotonin transporter occupancy following escalating dosing regimens of (R)‐(–)‐O‐desmethylvenlafaxine and racemic O‐desmethylvenlafaxine

SEP‐227162 [R(–)‐O‐desmethylvenlafaxine] is an enantiomer of the venlafaxine metabolite O‐desmethylvenlafaxine (ODV, Pristiq?, Wyeth). This study compared the serotonin transporter (SERT) occupancy achieved by SEP‐227162 and ODV, at daily doses of 25, 50, 100, and 150 mg using [¹¹C]DASB positron emission tomography (PET). Sixteen healthy male subjects participated in one of four dose groups (N = 4 per group) during which they were administered two doses of the study drug (SEP‐227162 or ODV). For each study drug, total daily doses of 25, 50, 100, and150 mg were studied. Subjects underwent three PET scans with [¹¹C]DASB. A baseline, off‐medication, scan was performed prior to dosing and a [¹¹C]DASB PET scan was performed after 72 hr at each dose level. [¹¹C]DASB binding potential (BP_ND) was calculated using the simplified reference tissue method. SERT occupancy was calculated as the change in BP_ND (ΔBP_ND) from baseline scan to the on‐medication scan relative to the baseline BP_ND value. SEP‐227162 and ODV significantly reduced regional distribution volumes and region BP_ND values in a dose‐dependent manner. Across all doses ODV produced significantly greater SERT occupancy than SEP‐227162 (ANOVA F = 21.8, df = 1,23, p < .001). The total daily dose required to provide 50% SERT occupancy was 24.8 mg for SEP‐227162 and 14.4 mg for ODV. In vitro data suggests a ratio of 3.3:1 for binding at human SERT for SEP‐227162 relative to ODV. Our study suggests a ratio of 1.7:1, highlighting the value of in vivo imaging in the drug development process.     

Characterization of the novel GlyT1 PET tracer [18F]MK‐6577 in humans

Decreased glutamatergic neurotransmission is hypothesized to be involved in the pathophysiology of schizophrenia. Inhibition of glycine transporter Type‐1 (GlyT1) reuptake is expected to increase the glutamatergic neurotransmission and may serve as treatment for cognitive and negative symptoms of schizophrenia. In this article, we present human data from a novel GlyT1 PET tracer, [¹⁸F]MK‐6577. In the process of developing a GlyT1 inhibitor therapeutic, a PET tracer can assist in determining the dose with a high probability of sufficiently testing the mechanism of action. This article reports the human PET studies with [¹⁸F]MK‐6577 for measuring GlyT1 receptor availability at baseline in normal human subjects and occupancy with a GlyT1 inhibitor, MK‐2637. Studies were also performed to measure radiation burden and the baseline test‐retest (T‐RT) variability of the tracer. The effective dose from sequential whole‐body dosimetry scans in three male subjects was estimated to be 24.5 ± 2.9 µSV/MBq (mean ± SD). The time–activity curves from T‐RT scans modeled satisfactorily using a two tissue compartmental model. The tracer uptake was highest in the pons (V_T = 6.7 ± 0.9, BP_ND = 4.1 ± 0.43) and lowest in the cortex (V_T = 2.1 ± 0.5, BP_ND = 0.60 ± 0.23). V_T T‐RT variability measured in three subjects was <12% on average. The occupancy scans performed in a cohort of 15 subjects indicated absence of a reference region. The in vivo potency (Occ₅₀) of MK‐2637 was determined using two methods: A: Lassen plot with a population input function (Occ₅₀ = 106 nM, SE = 20 nM) and B: pseudo reference tissue model using cortex as the pseudo reference region (Occ₅₀ = 141 nM, SE = 21 nM). Synapse 69:33–40, 2015. © 2014 Wiley Periodicals, Inc.     

Imaging histamine H3 receptors with [18F]FMH3: Test–retest and occupancy studies in the non‐human primate

A positron emission tomography (PET) radioligand, [¹⁸F]FMH3, has been developed to interrogate histamine receptor subtype 3 (H3R), where dysfunction at this site is linked with obesity, sleep abnormality, and cognitive disorders. [¹⁸F]FMH3 was evaluated for imaging central H3R sites in non‐human primates through test–retest (TRT) and dose‐receptor occupancy studies with two selective H3R antagonists in order to support clinical investigations. Two adult female baboons underwent [¹⁸F]FMH3 PET brain scans in the HR+, at repeated baseline (n = 7) and following administration of escalating doses of ABT‐239 (0.003–0.1m/kg, n = 4) and ciproxifan (0.5–2.1 mg/kg, n = 7). Volume of distribution (V_T) in brain regions was estimated using the 2‐tissue compartment model. TRT variability of V_T across repeated baseline scans was reported as % coefficient of variation (COV). ABT‐239 and ciproxifan occupancy at H3R was estimated using the occupancy plot, and the relationship of occupancy with dose and plasma levels was determined. In baboons, distribution of [¹⁸F]FMH3 was high in the striatum, intermediate in cortical regions, and low in the brain stem. COV of baseline V_T was 7.0 ± 3.5%, averaged across regions and animals. Dose‐dependent effects of ABT‐239 and ciproxifan measured the brain. ED50 and EC50, respectively, were 0.011 mg/kg and 0.942 ng/ml for ABT‐239 and 0.73 mg/kg and 208.3 ng/ml for ciproxifan. [¹⁸F]FMH3 demonstrated high TRT reliability and can be used to measure occupancy of H3R‐targeted drugs. Validation in non‐human primates support [¹⁸F]FMH3 PET studies toward clinical investigations of H3R.     

Activation of alpha‐1 adrenergic receptors increases cytosolic calcium in neurones of the paraventricular nucleus of the hypothalamus

Norepinephrine (NE) activates adrenergic receptors (ARs) in the hypothalamic paraventricular nucleus (PVN) to increase excitatory currents, depolarise neurones and, ultimately, augment neuro‐sympathetic and endocrine output. Such cellular events are known to potentiate intracellular calcium ([Ca²⁺]_i); however, the role of NE with respect to modulating [Ca²⁺]_i in PVN neurones and the mechanisms by which this may occur remain unclear. We evaluated the effects of NE on [Ca²⁺]_i of acutely isolated PVN neurones using Fura‐2 imaging. NE induced a slow increase in [Ca²⁺]_i compared to artificial cerebrospinal fluid vehicle. NE‐induced Ca²⁺ elevations were mimicked by the α₁‐AR agonist phenylephrine (PE) but not by α₂‐AR agonist clonidine (CLON). NE and PE but not CLON also increased the overall number of neurones that increase [Ca²⁺]_i (ie, responders). Elimination of extracellular Ca²⁺ or intracellular endoplasmic reticulum Ca²⁺ stores abolished the increase in [Ca²⁺]_i and reduced responders. Blockade of voltage‐dependent Ca²⁺ channels abolished the α₁‐AR induced increase in [Ca²⁺]_i and number of responders, as did inhibition of phospholipase C inhibitor, protein kinase C and inositol triphosphate receptors. Spontaneous phasic Ca²⁺ events, however, were not altered by NE, PE or CLON. Repeated K⁺‐induced membrane depolarisation produced repetitive [Ca²⁺]_i elevations. NE and PE increased baseline Ca²⁺, whereas NE decreased the peak amplitude. CLON also decreased peak amplitude but did not affect baseline [Ca²⁺]_i. Taken together, these data suggest receptor‐specific influence of α₁ and α₂ receptors on the various modes of calcium entry in PVN neurones. They further suggest Ca²⁺ increase via α₁‐ARs is co‐dependent on extracellular Ca²⁺ influx and intracellular Ca²⁺ release, possibly via a phospholipase C inhibitor‐mediated signalling cascade.     

α2‐Adrenergic drugs modulate the binding of [18F]fallypride to dopamine D2/3 receptors in striatum of living mouse

Aim. To test for α₂ adrenergic modulation of dopamine D_2/3 receptor availability in striatum of living mice using the high‐affinity ligand [¹⁸F]fallypride and microPET. Methods. Groups of anesthetized mice were pretreated with saline, the α₂‐agonist clonidine (1 mg/kg), and the α₂‐antagonists RX821002 (1 mg/kg) and yohimbine (1 mg/kg). Dynamic microPET recordings lasting 120 min were then initiated upon i.v. tracer injection of [¹⁸F]fallypride. Parametric maps of [¹⁸F]fallypride binding potential (BP_ND) were calculated using the Logan method, with cerebellum serving as the reference region. Results. Mean striatal [¹⁸F]fallypride BP_ND was 10.6 ± 1.7 in the saline control animals, 8.9 ± 1.7 (?16%; P < 0.05) in the RX821002 group, 8.3 ± 2.6 (?22%; P < 0.05) in the yohimbine group and 10.3 ± 2.2 (n.s.) in the clonidine group. Conclusions. These findings are consistent with a tonic inhibition of dopamine release by α₂ adrenergic receptors, such that α₂ blockade increased the competition from endogenous dopamine at D_2/3 receptors, thus reducing the [¹⁸F]fallypride BP_ND by about 20%. Absent effects of clonidine suggest a ceiling effect in the tonic inhibition of dopamine release. This in vivo PET evidence for α₂/dopaminergic interaction may be relevant to putative actions of atypical antipsychotic medications via adrenergic receptors. Synapse, 2010. © 2010 Wiley‐Liss, Inc.     

[18F]Nifene test–retest reproducibility in first‐in‐human imaging of α4β2* nicotinic acetylcholine receptors

The aim of this study was to examine the suitability of [¹⁸ F]nifene, a novel α4β2* nicotinic acetylcholine receptor (nAChR) radiotracer, for in vivo brain imaging in a first‐in‐human study. Methods : Eight healthy subjects (4 M,4 F;21–69,44 ± 21 yrs) underwent a [¹⁸F]nifene positron emission tomography scan (200 ± 3.7 MBq), and seven underwent a second scan within 58 ± 31 days. Regional estimates of DVR were measured using the multilinear reference tissue model (MRTM2) with the corpus callosum as reference region. DVR reproducibility was evaluated with test–retest variability (TRV) and intraclass correlation coefficient (ICC). Results : The DVR ranged from 1.3 to 2.5 across brain regions with a TRV of 0–7%, and did not demonstrate a systematic difference between test and retest. The ICCs ranged from 0.2 to 0.9. DVR estimates were stable after 40 min. Conclusion : The binding profile and tracer kinetics of [¹⁸F]nifene make it a promising α4β2* nAChR radiotracer for scientific research in humans, with reliable DVR test–retest reproducibility.