Human MT Research Articles

Mapping the Melatonin Receptor. 7. Subtype Selective Ligands Based on β-Substituted N-Acyl-5-methoxytryptamines and β-Substituted N-Acyl-5-methoxy-1-methyltryptamines

A series of beta-substituted and beta,beta-disubstituted N-acyl 5-methoxy-1-methyltryptamines and 5-methoxytryptamines have been prepared as melatonin analogues to investigate the nature of the binding site of the melatonin receptor. The affinity of analogues was determined in a radioligand binding assay using cloned human MT(1) and MT(2) receptor subtypes expressed in NIH 3T3 cells. Agonist and antagonist potency of all analogues was measured using the pigment aggregation response of a clonal line of Xenopus laevis melanophores. beta-Methylmelatonin (17a) and beta,beta-dimethylmelatonin (17b), though showing a slight decrease in binding at human receptors, show an increase in potency on Xenopus. N-Butanoyl 5-methoxy-1-methyl-beta,beta-trimethylenetryptamine (12c) is an antagonist at human MT(1) receptors but an agonist at MT(2), while N-butanoyl 5-methoxy-1-methyl-beta,beta-tetramethylenetryptamine (13c) is an antagonist at MT(1) but had no action at MT(2) and is one of the first examples of an MT(1) selective antagonist.

Sensitivity to optic flow in human cortical areas MT and MST

The macaque V5/MT complex comprises several sub-regions but little is known of their human homologues. We examined human V5/MT with fMRI in terms of specificity to optic flow stimuli, a key characteristic of macaque MST. Stimuli were large fields of moving dots, forming coherent global flow patterns. Random motion was used as a control. Retinotopic mapping was also conducted. The previously suggested existence of at least two distinct sub-regions, MT and MST, within the V5/MT complex was confirmed. Human MT is activated about equally by all moving dot patterns, including random motion, suggesting that it has little sensitivity to global flow structure. As previously described, this region shows strong signs of retinotopic organization and is only weakly activated by stimuli confined to the ipsilateral hemifield. In human MST, located immediately anterior to MT and strongly driven by ipsilateral stimuli, activation varies markedly with optic flow structure. The strongest activation is produced by complex flow that contains multiple flow components (expansion, contraction and rotation). Single components produce rather less response, while rigid translation and random motion produce less still. The results suggest that human MST is strongly specialized for encoding global flow properties, while human MT is less so.

Cross-feature spread of global attentional modulation in human area MT+

Feature-based attention affects the processing of the selected feature throughout the visual field. Here, we show that such global attentional modulation is not restricted to the attended feature but spreads to task-irrelevant features that are bound to the attended one. Attention to a color in one of the visual hemifields affected the processing of task-irrelevant motion in the other hemifield when it was associated with a stimulus that shared the attended color. This cross-feature global attentional selection increased the duration of the motion aftereffect and the strength of functional magnetic resonance imaging responses in the motion-sensitive area MT+, evoked by the task-irrelevant motion. These findings imply that features belonging to the same object are bound and selected jointly even outside the focus of attention.

Contrast Adaptation and Representation in Human Early Visual Cortex

The human visual system can distinguish variations in image contrast over a much larger range than measurements of the static relationship between contrast and response in visual cortex would suggest. This discrepancy may be explained if adaptation serves to re-center contrast response functions around the ambient contrast, yet experiments on humans have yet to report such an effect. By using event-related fMRI and a data-driven analysis approach, we found that contrast response functions in V1, V2, and V3 shift to approximately center on the adapting contrast. Furthermore, we discovered that, unlike earlier areas, human V4 (hV4) responds positively to contrast changes, whether increments or decrements, suggesting that hV4 does not faithfully represent contrast, but instead responds to salient changes. These findings suggest that the visual system discounts slow uninformative changes in contrast with adaptation, yet remains exquisitely sensitive to changes that may signal important events in the environment.

Specific retinotopically based magnocellular impairment in a patient with medial visual dorsal stream damage

We report here retinotopically based magnocellular deficits in a patient with a unilateral parieto-occipital lesion. We applied convergent methodologies to study his dorsal stream processing, using psychophysics as well as structural and functional imaging. Using standard perimetry we found deficits involving the periphery of the left inferior quadrant abutting the horizontal meridian, suggesting damage of dorsal retinotopic representations beyond V1. Retinotopic damage was much more extensive when probed with frequency-doubling based contrast sensitivity measurements, which isolate processing within the magnocellular pathway: sensitivity losses now encroached on the visual central representation and did not respect the horizontal meridian, suggesting further damage to dorsal stream retinotopic areas that contain full hemi-field representations, such as human V3A or V6. Functional imaging revealed normal responses of human MT + to motion contrast. Taken together, these findings are consistent with a recent proposal of two distinct magnocellular dorsal stream pathways: a latero-dorsal pathway passing to MT + and concerned with the processing of coherent motion, and a medio-dorsal pathway that routes information from V3A to the human homologue of V6. Anatomical evidence was consistent with sparing of the latero-dorsal pathway in our patient, and was corroborated by his normal performance in speed, direction discrimination and motion coherence tasks with 2D and 3D objects. His pattern of dysfunction suggests damage only to the medio-dorsal pathway, an inference that is consistent with structural imaging data, which revealed a lesion encompassing the right parieto-occipital sulcus.

Identification of WIN55212-3 as a competitive neutral antagonist of the human cannabinoid CB2 receptor.

1. Several G protein-coupled receptors (GPCRs), including cannabinoid CB(1) and CB(2) receptors, show constitutive activity under heterologous expression. Such a tonic response is generated in the absence of an activating ligand, and can be inhibited by inverse agonists. Neutral antagonists, however, are silent at such receptors, but can reverse both agonist and inverse agonist responses. To date, no neutral antagonist for the CB(2) receptor has been reported. 2. Here, by monitoring receptor-dependent G protein activation, we demonstrate that WIN55212-3 acts as a neutral antagonist at the human CB(2) (hCB(2)) receptor. WIN55212-3 alone, at concentrations </=10(-4) M, behaved as a silent ligand exhibiting no agonist or inverse agonist activity. However, WIN55212-3 competitively antagonized cannabinoid agonist CP-55,940-stimulated responses (pA(2) 6.1). Importantly, the inverse agonism evoked by SR144528 in hCB(2) was dose-dependently reversed by WIN55212-3 (pEC(50) 5.3+/-0.2), indicating true neutral antagonist behavior. 3. Furthermore, WIN55212-3 also antagonized CB(1) receptor signaling in a competitive manner (pA(2) 5.6), but behaved as a partial inverse agonist (pIC(50) 5.5+/-0.1) at the constitutively active human CB(1). 4. Additionally, WIN55212-3 antagonized signaling of the human melatonin MT(1) receptor, with modest activity at the human muscarinic M4 receptor, but it was inactive towards several other GPCRs. 5. These data identify WIN55212-3 as a true neutral hCB(2) receptor antagonist. WIN55212-3 offers a valuable tool for further characterization of ligand activities at the CB(2) receptor and may serve as a lead compound in further efforts to develop more potent and selective neutral CB(2) receptor antagonists.

Analysis of Structure−Activity Relationships for MT2 Selective Antagonists by Melatonin MT1 and MT2 Receptor Models

Three-dimensional homology models of human MT(1) and MT(2) melatonin receptors were built with the aim to investigate the structure-activity relationships (SARs) of MT(2) selective antagonists. A common interaction pattern was proposed for a series of structurally different MT(2) selective antagonists, which were positioned within the binding site by docking and simulated annealing. The proposed antagonist binding mode to the MT(2) receptor is characterized by the accommodation of the out-of-plane substituents in a hydrophobic pocket, which resulted as being fundamental for the explanation of the antagonist behavior and the MT(2) receptor selectivity. Moreover, to assess the ability of the MT(2) receptor model to reproduce the SARs of MT(2) antagonists, three new derivatives of the MT(2) selective antagonist N-[1-(4-chloro-benzyl)-4-methoxy-1H-indol-2-ylmethyl]-propionamide (7) were synthesized and tested for their receptor affinity and intrinsic activity. These compounds were docked into the MT(2) receptor model and were submitted to molecular dynamics studies, providing results in qualitative agreement with the experimental data. These results confirm the importance of the out-of-plane group in receptor binding and selectivity and provide a partial validation of the proposed G protein-coupled receptor model.

Specializations for Chromatic and Temporal Signals in Human Visual Cortex

Neurological case studies and qualitative measurements suggest that regions within human extrastriate cortex are specialized for different perceptual functions, including color. However, there are few quantitative measurements of human extrastriate color specializations. We studied the chromatic and temporal responses in several different clusters of human visual field maps using functional magnetic resonance imaging. Contrast response functions were measured for luminance [(L + M)-cone], red-green [(L - M)-cone] and blue-yellow (S-cone) modulations at various temporal frequencies. In primary visual cortex (V1), temporal responsivities to luminance and red-green modulations are approximately constant up to 10 Hz, but responsivities to blue-yellow modulations decrease significantly. In ventral occipital cortex (VO), all colors elicit strong responses, and, for each color, low temporal frequency modulations are more effective than high temporal frequency modulations. Hence, VO represents the full range of color information but does not respond well to rapid modulations. Conversely, in human motion-selective cortex (MT+) and V3A, blue-yellow modulations elicit very weak responses, whereas luminance and red-green high temporal frequency modulations are equally or more effective than low temporal frequency modulations. Hence, these dorsal occipital regions respond well to rapid modulations, but not all color information is represented. Similar to human motion perception, MT+ and V3A respond powerfully to all temporal frequencies but only to some colors. Similar to human color perception, VO responds powerfully to all colors but only to relatively low temporal frequencies.

The Human MT1 Melatonin Receptor Stimulates cAMP Production in the Human Neuroblastoma Cell Line SH‐SY5Y Cells Via a Calcium‐Calmodulin Signal Transduction Pathway

Melatonin regulates circadian and seasonal physiology via melatonin receptors expressed in the brain. However, little is known about the signal transduction mechanisms that mediate the action of melatonin in neuronal cells. To begin to address this issue, we expressed the human MT(1) receptor in the human neuroblastoma SH-SY5Y cell line. In this cell line, melatonin acutely stimulated cAMP synthesis through a calcium-calmodulin dependent pathway. This stimulatory effect was independent of an interaction with G(i) or G(s) G proteins and dependent upon internal calcium stores. Melatonin also potentiated forskolin-activated cAMP synthesis. Differentiation of the neuroblastoma cells with retinoic acid to the neuronal phenotype did not alter the ability of melatonin to acutely stimulate cAMP. These data may be relevant to the neuronal action of melatonin and highlight the importance of the cellular context of expression of melatonin and other G protein-coupled receptors.

Visual Mental Imagery Induces Retinotopically Organized Activation of Early Visual Areas

There is a long-standing debate as to whether visual mental imagery relies entirely on symbolic (language-like) representations or also relies on depictive (picture-like) representations. We sought to discover whether visual mental imagery could evoke cortical activity with precise visual field topography (retinotopy). Participants received three conditions: the perception condition consisted of a standard retinotopic mapping procedure, where two flickering checkerboard wedges rotated around a central fixation point. The imagery and attention conditions consisted of the same stimulus, but only the outer arcs of the wedges were visible. During imagery, participants mentally reproduced the stimulus wedges, using the stimulus arcs as a guide. The attention condition required either distributed attention or focused attention to where the stimulus wedges would have been. Event-related analysis revealed that the imagery (greater than either form of attention) retinotopic maps were similar to the perception maps. Moreover, blocked analysis revealed similar perception and imagery effects in human motion processing region MT+. These results support the depictive view of visual mental imagery.

( R)-2-(4-Phenylbutyl)dihydrobenzofuran derivatives as melatoninergic agents

( R)-2-(4-Phenylbutyl)dihydrobenzofuran derivatives (e.g., 3 and 4) were synthesized as novel melatoninergic ligands with significantly lower vasoconstrictive activity in vitro in the rat tail artery. Binding affinity assays were performed on cloned human MT 1 and MT 2 receptors stably expressed in NIH3T3 cells.

Task-dependent activation latency in human visual extrastriate cortex

Event related potentials (ERPs) were recorded from subjects who had to perform either an identification task or a simple detection task on moving visual stimuli. Results showed that the amplitude of the so-called visual “N1” component was larger for identification than for mere detection, replicating previous data obtained with static stimuli. However, we also found that: (i) the onset, peak and offset latencies of the visual N1 to dynamic stimuli were significantly earlier in the detection task than in the identification task, and (ii) in both conditions, the coordinates of the equivalent current dipoles best explaining the visual N1 component were consistent with those of the human motion visual area MT+/V5 in the extrastriate cortex. Altogether, these results indicate that dynamic stimuli may activate (at least partly) different pathways and processes in extrastriate cortex according to the nature of the task required on these stimuli.

Localization of the Human Cortical Visual Area MT Based on Computer Aided Histological Analysis

We describe human area MT histologically based on the observer independent analysis of cortical myeloarchiteture, multiple complementary staining techniques and 3-D reconstruction. The topography of an architectonic field that presented constant structural characteristics across specimens was studied in relation to the sulcal geography of the occipito-temporal region. Objective and semi-automated analysis of local microstructure revealed a distinct cortical architecture and matched topographically the localization of MT derived from functional imaging. MT was localized by the histotopographic method in relation to definite macroscopic landmarks. This study demonstrates a new set of distinguishing architectonic features of human MT that permit localization on structural grounds and suggests that the characteristic laminar structure of this area may be related to its unique pattern of connections and to its role in visual perception.

Neurochemical properties of ramelteon (TAK-375), a selective MT 1/MT 2 receptor agonist

Ramelteon (TAK-375) is a novel melatonin receptor agonist currently under investigation for the treatment of insomnia. This study describes the neurochemical and receptor binding characteristics of ramelteon in vitro. Ramelteon showed very high affinity for human MT 1 (Mel 1a) and MT 2 (Mel 1b) receptors (expressed in Chinese hamster ovary [CHO] cells), and chick forebrain melatonin receptors (consisting of Mel 1a and Mel 1c receptors) with K i values of 14.0, 112, and 23.1 pM, respectively, making the affinities of ramelteon for these receptors 3–16 times higher than those of melatonin. The affinity of ramelteon for hamster brain MT 3 binding sites was extremely weak ( K i: 2.65 μM) compared to melatonin's affinity for the MT 3 binding site ( K i: 24.1 nM). In addition, ramelteon showed no measurable affinity for a large number of ligand binding sites (including benzodiazepine receptors, dopamine receptors, opiate receptors, ion channels, and transporters) and no effect on the activity of various enzymes. Ramelteon inhibited forskolin-stimulated cAMP production in the CHO cells that express the human MT 1 or MT 2 receptors. Taken together, these results indicate that ramelteon is a potent and highly selective agonist of MT 1/MT 2 melatonin receptors.

Direct current stimulation over MT+/V5 modulates motion aftereffect in humans

While there is strong evidence for the central role of the human MT+/V5 in motion processing, its involvement in motion adaptation is still the subject of debate. We used transcranial direct current stimulation (tDCS) to test whether MT+/V5 is part of the neural network involved in the long-term adaptation-induced motion after-effect in humans. It was found that both cathodal and anodal stimulation over MT+/V5 resulted in a significant reduction of the perceived motion after-effect duration, but had no effect on performance in a luminance-change-detection task used to determine attentional load during adaptation. Our control experiment excluded the possibility that the observed MT+/V5 stimulation effects were due to a diffused modulation of the early cortical areas, i.e. by the stimulation applied over MT+/V5. These results provide evidence that external modulation of neural excitability in human MT+/V5 affects the strength of perceived motion after-effect and support the involvement of MT+/V5 in motion adaptation processes.

Knock-down of RGS 4 and β tubulin in CHO cells expressing the human MT 1 melatonin receptor prevents melatonin-induced receptor desensitization

Previously, it has been shown that chronic melatonin exposure in MT1-CHO cells results in receptor desensitization while at the same time producing drastic morphological changes. The addition of a depolymerizing agent during the melatonin pretreatment period prevents MT 1 receptor desensitization and the changes in cellular morphology. The lack of morphological change in the presence of a depolymerizing agent is easily explained by the inability of the microtubules to polymerize, however, the prevention of receptor desensitization is a little more complex and may involve G-protein activation. The goal of this study was to determine whether melatonin-induced MT 1 receptor desensitization is regulated by proteins known to regulate G-protein activation states, β-tubulin and RGS 4,using anti sense knockdown approaches. The expression of RGS 4 mRNA in CHO cells was confirmed using RT PCR and successful knockdown of each was confirmed by western blot analysis or quantitative PCR. Pretreatment of MT1-CHO cells, transfected with the nonsense probes and exposed to melatonin, resulted in a desensitization of the receptor, an increase in forskolin-induced cAMP accumulation, an increase in 2-[ 125I]-iodomelatonin binding and no change in the affinity of melatonin for the MT 1 receptor. However, knockdown of either β−tubulin or RGS 4 in MT1-CHO cells followed by pretreatment with melatonin attenuated the desensitization of melatonin receptors, decreased total 2-[ 125I]-iodomelatonin binding, and did not affect neither the forskolin response nor the affinity of melatonin for the MT 1 receptor. Perhaps RGS 4 and β-tubulin modulate Gα-GDP and Gα-GTP states thus modulating MT 1 melatonin receptor function.

Chronobiotic activity of N-[2-(2,7-dimethoxyfluoren-9-yl)ethyl]-propanamide. Synthesis and melatonergic pharmacology of fluoren-9-ylethyl amides

A series of fluoren-9-yl ethyl amides ( 2) were synthesized and evaluated for human melatonin MT 1 and MT 2 receptor binding. N-[2-(2,7-dimethoxyfluoren-9-yl)ethyl]propanamide ( 2b) was selected and evaluated in functional assays measuring intrinsic activity at the human MT 1 and MT 2 receptors and demonstrated full agonism at both receptors. The chronobiotic properties of 2b were demonstrated in both acute and chronic rat models where 2b produced an acute phase advance of 32 min at 1 mg/kg and chronically entrained free-running rats with a mean effective dose of 0.23 mg/kg. Compound 2b is significantly less efficacious than melatonin in constricting human coronary artery.

Design and Synthesis of Benzoxazole Derivatives as Novel Melatoninergic Ligands.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Subregions of human MT complex revealed by comparative MEG and direct electrocorticographic recordings

Objective: To locate the visual motion complex (MT+) and study its response properties in an epilepsy surgery patient. Methods: A 17-year-old epilepsy patient underwent invasive monitoring with subdural electrodes in the right temporo-parieto-occipital area. MT+ was investigated by cortical electric stimulation and by epicortical visual evoked potentials time-locked to motion onset of sinusoidal gratings (motion VEP). Motion-related visual evoked magnetic field (motion VEF) was also recorded before the electrode implantation to complement the invasive recording. Results: Motion VEPs revealed two subregions within MT+, generating early and late potentials respectively. The early activity with a peak around 130 ms was localized at a single electrode situated immediately caudal to the initial portion of the ascending limb of the superior temporal sulcus (AL-STS). The late activity, peaking at 242–274 ms, was located ventro-rostrally over three electrodes. Among the four electrodes with motion VEPs, cortical stimulation at the most caudal pair elicited motion-in-depth perception involving the whole visual field. In addition to two subregions revealed on the gyral crown, magnetoencephalography (MEG) demonstrated another subregion with a late motion VEF in AL-STS immediately rostral to the electrode with the early motion VEP. Conclusions: In combination with MEG recording, the present invasive exploration demonstrated human MT+ in a focal area of the temporo-parieto-occipital junction and delineated possible three subregions as indicated by the different latencies and distributions of the motion VEP/VEFs. Significance: Comparative MEG and direct electrocorticographic recordings delineated possible subregions within the human MT complex.

Exploring QSAR of melatonin receptor ligand benzofuran derivatives using E-state index

Considering the recent challenge to the medicinal chemists for the development of selective melatonin receptor ligands, an attempt has been made to explore physicochemical requirements of benzofuran derivatives for binding with human MT 1 and MT 2 receptor subtypes and also to explore selectivity requirements. In this study, E-states of different common atoms of the molecules (calculated according to Kier and Hall) and physicochemical parameters (partition coefficient and molar refractivity) were used as independent variables along with suitable dummy parameters. The best equation describing MT 1 binding affinity [ n=34, Q 2=0.670, R a 2=0.790, R 2=0.822, R=0.907, s=0.609, F=25.8 ( df 5, 28)] suggests that the binding affinity decreases as the value of n (number of CH 2 spacer beside R 2) increases while it increases with rise in electrotopological state values of different atoms of the benzofuran ring. Again, presence of methoxy group at R 1 and hydrogen, unsubstituted phenyl or fluoro-substituted phenyl group at R 2 is conducive to the MT 1 binding affinity. The binding affinity decreases if furyl substitution at R 3 position is present. The best equation describing MT 2 binding affinity [ n=34, Q 2=0.602, R a 2=0.755, R 2=0.792, R=0.890, s=0.584, F=21.3 ( df 5, 28)] shows that the MT 2 binding affinity depends on the similar factors as described for MT 1 binding affinity; however, the contributions of the factors for the two affinities are different to some extent as evidenced from the regression coefficients. Among the selectivity relations, the best equation [ n=33, Q 2=0.496, R a 2=0.681, R 2=0.721, R=0.849, s=0.458, F=18.1 ( df 4, 28)] suggests that MT 2 binding increases with increase in value of n, presence of methoxy group at R 1, and E-state values of different atoms of the benzofuran ring, while it decreases in presence of furyl group at R 3 position.