Localization of Melatonin Receptor 1 in Mouse Retina and Its Role in the Circadian Regulation of the Electroretinogram and Dopamine Levels

Melatonin modulates many important functions within the eye by interacting with a family of G-protein-coupled receptors that are negatively coupled with adenylate cyclase. In the mouse, Melatonin Receptors type 1 (MT1) mRNAs have been localized to photoreceptors, inner retinal neurons, and ganglion cells, thus suggesting that MT1 receptors may play an important role in retinal physiology. Indeed, we have recently reported that absence of the MT1 receptors has a dramatic effect on the regulation of the daily rhythm in visual processing, and on retinal cell viability during aging. We have also shown that removal of MT1 receptors leads to a small (3–4 mmHg) increase in the level of the intraocular pressure during the night and to a significant loss (25–30%) in the number of cells within the retinal ganglion cell layer during aging. In the present study we investigated the cellular distribution in the C3H/f+/+ mouse retina of MT1 receptors using a newly developed MT1 receptor antibody, and then we determined the role that MT1 signaling plays in the circadian regulation of the mouse electroretinogram, and in the retinal dopaminergic system. Our data indicate that MT1 receptor immunoreactivity is present in many retinal cell types, and in particular, on rod and cone photoreceptors and on intrinsically photosensitive ganglion cells (ipRGCs). MT1 signaling is necessary for the circadian rhythm in the photopic ERG, but not for the circadian rhythm in the retinal dopaminergic system. Finally our data suggest that the circadian regulation of dopamine turnover does not drive the photopic ERG rhythm

Crizotinib in MET-Deregulated or ROS1-Rearranged Pretreated Non-Small Cell Lung Cancer (METROS): A Phase II, Prospective, Multicenter, Two-Arms Trial.

PURPOSE: MET-deregulated NSCLC represents an urgent clinical need because of unfavorable prognosis and lack of specific therapies. Although recent studies have suggested a potential role for crizotinib in patients harboring MET amplification or exon 14 mutations, no conclusive data are currently available. This study aimed at investigating activity of crizotinib in patients harboring MET or ROS1 alterations. PATIENTS AND METHODS: Patients with pretreated advanced NSCLC and evidence of ROS1 rearrangements (cohort A) or MET deregulation (amplification, ratio MET/CEP7 >2.2 or MET exon 14 mutations, cohort B) were treated with crizotinib 250 mg twice daily orally. The coprimary endpoint was objective response rate in the two cohorts. RESULTS: From December 2014 to March 2017, 505 patients were screened and a total of 52 patients (26 patients per cohort) were enrolled onto the study. At data cutoff of September 2017, in cohort A, objective response rate was 65%, and median progression-free survival and overall survival were 22.8 months [95% confidence interval (CI) 15.2-30.3] and not reached, respectively. In cohort B, objective response rate was 27%, median progression-free survival was 4.4 months (95% CI 3.0-5.8), and overall survival was 5.4 months (95% CI, 4.2-6.5). No difference in any clinical endpoint was observed between MET-amplified and exon 14-mutated patients. No response was observed among the 5 patients with cooccurrence of a second gene alteration. No unexpected toxicity was observed in both cohorts. CONCLUSIONS: Crizotinib induces response in a fraction of MET-deregulated NSCLC. Additional studies and innovative therapies are urgently needed

Age-Related Changes in the Daily Rhythm of Photoreceptor Functioning and Circuitry in a Melatonin-Proficient Mouse Strain

Retinal melatonin is involved in the modulation of many important retinal functions. Our previous studies have shown that the viability of photoreceptors and ganglion cells is reduced during aging in mice that lack melatonin receptor type 1. This demonstrates that melatonin signaling is important for the survival of retinal neurons. In the present study, we investigate the effects of aging on photoreceptor physiology and retinal organization in CH3-f+/+ mice, a melatonin proficient mouse strain. Our data indicate that the amplitude of the a and b waves of the scotopic and photopic electroretinogram decreases with age. Moreover, the daily rhythm in the amplitude of the a- and b- waves is lost during the aging process. Similarly, the scotopic threshold response is significantly affected by aging, but only when it is measured during the night. Interestingly, the changes observed in the ERGs are not paralleled by relevant changes in retinal morphological features, and administration of exogenous melatonin does not affect the ERGs in C3H-f+/+ at 12 months of age. This suggests that the responsiveness of the photoreceptors to exogenous melatonin is reduced during aging

COMPARISON OF THE EFFECTS OF BARBEXACLONE AND PHENOBARBITAL ON EPILEPTIC PATIENTS

This study evaluated the effects of barbexaclone on the frequency of seizures in a group of 40 epileptics, previously treated with phenobarbital. The effect of barbexaclone compared with phenobarbital on vigilance was assessed by means of the Digit Symbol Substitution Test, visuomotor reaction times in lateralized vision, and a subjective scale. After four and eight months of barbexaclone therapy, serum phenobarbital levels were substantially unchanged, while the frequency of seizures was reduced by at least half in 25% of the patients examined. In the level of vigilance, patients reported less drowsiness, confirmed by improvements in performance of the Digit Symbol Substitution Test. No specific correlation with reaction times was observed, howeve

Effect of aging on the STR.

<p>(A) No differences were observed at ZT6 in the STR among young and old mice (t-tests, P>0.5 in all cases). Black circles, white circles, and black triangles indicate 3-, 6-, and 12-month-old mice, respectively. Each point represents the mean ± SEM, N = 6–8 for each point. (B) At ZT18 the STR significantly increased (−4.60 log log cd*s/m² vs −3.60 log cd*s/m²n) in 12-month-old mice (t-tests, P<0.05). Figures C through E show representative traces of the ERG at the different ages (C = 3 months; D = 6 months; E = 12 months).</p

Effect of administration of exogenous melatonin on the scotopic ERG at different ages.

<p>Melatonin injection (1 mg/Kg) increased the amplitude of the a (A, B) and b wave (D, E) of the scotopic ERG in 3- and 6-month-old mice (Two-way ANOVA P<0.01, followed by Tukey tests, P<0.05) with respect to the values obtained in control mice. The same dose did not increase the amplitude of the a and b wave in 12 months old mice (Two-way ANOVA, P>0.01, C, F). Melatonin injection (0.1 mg/Kg) increased the amplitude of the a and b waves of the scotopic ERG in 3-month-old mice (Two-way ANOVA, P<0.01, followed by Tukey tests, P<0.05 A, D), but not in 6 month-old mice (Two-way ANOVA, P>0.05; B, E, C, F). White circles indicate control groups; black triangles indicate melatonin 0.01 mg/kg; white squares indicate melatonin 0.1 mg/kg; and black circles indicate melatonin 1 mg/kg. Each value represents the mean ± SEM, N = 5–6 for each point.</p

Effect of aging on the photopic ERG and its daily rhythm.

<p>(A) The amplitude of b wave of the photopic ERG at ZT6 was not affected by age at ZT6 (Two-way ANOVA, P>0.1). (B) It showed a significant decrease at ZT18 (Two-way ANOVA, P, 0.01, followed by Tukey tests, P<0.05) A significant difference in the amplitude of the b wave between ZT6 and ZT18 was only detected in 3-month-old mice (Two-way ANOVA, P>0.1 followed by Tukey tests, P<0.05). Closed circles, open circles, and closed triangle indicate 3-, 6- and 12-month-old mice, respectively. Each data point represents 4 to 8 animals, and the mean ± SEM, where N = 6–8 for each point. Figures C through E show representative wave forms of each age group at ZT18 (C = 3 months; D = 6 months and E = 12 months).</p

Retinal organization in C3H-f+/+ at 12 months of age.

<p>TOTO-3 staining of nuclear layers (red in A and blue in B) reveals an intact retinal layering in C3H-f^+/+ control 12-month-old mice. (A) Rod bipolar cells labelled by protein kinase c (PKCα) antibodies (green) have normal morphology and layering. (B) PKCα antibodies show again RB (red) with normally distributed bassoon puncta (green) decorating their dendritic tips in the opl. (C) Rod bipolar cells (RB) (red) have dendritic tips associated to green puncta, representing kinesins II positive synaptic ribbons. Most puncta are appropriately confined in the opl, but some of them are displaced in the onl (arrows). (D) PKCα labelling of Rod bipolar cells (red) and PSD-95 (green). Most presynaptic endings of photoreceptors (labelled by PSD-95) are confined to the OPL, but a few are clearly seen in the onl (arrow). (E) Antibodies against Goα (green), specific for depolarizing bipolar cells, in combination with PKCα (red) allow visualization of rod bipolar cells (yellow-orange) and depolarizing cone bipolars (green, CB). Dendrites of both categories of cells are clearly visible (arrows). (F) Calbindin (red) in combination with neurofilament (green) antibodies show a normal pattern of staining of horizontal cell bodies (HC), their axonal endings (yellow profiles in the opl) and calbindin positive amacrine cells (AC). Neurofilament antibodies also stains ganglion cells (GC, arrow). (G) One tyrosine hydroxylase (TH) positive amacrine cell with the typical large size body (arrow) and main dendritic plexus in the outermost part of the ipl. (H) Antibodies against the enzyme glutamine synthase show the fine morphology of Müller glial cells (green). Anti-glial fibrillary acidic protein (GFAP) antibodies show astrocytes regularly placed at the innermost retinal margin (red, arrow). Scale bars are 20 μm. Ph: Photoreceptors; onl: outer nuclear; opl: outer plexiform; inl: inner nuclear; ipl: inner plexiform; gcl: ganglion cell layer.</p

MT₁ receptor immunoreactivity in the mouse retina.

<p>A band corresponding to the molecular weight of approximately 40 kDa was observed in western blotting of retinal extract from WT mice. No band was observed when the blot was treated with antibody pre-absorbed with the blocking peptide. A band of approximately 80 kDa was also present in retinal extract from WT mice. A very faint band around 80 kDa was also present the blot treated with the blocking peptide and in the blot of retinal extract from MT₁^−/− mice. α-Tubulin expression at the molecular weight of 53 kDa is also shown. Ab = Melatonin receptor 1 antibody; BP = blocking peptide. Similar results have been obtained for at least 3 independent samples for each experimental condition (Top panel). MT₁ immunoreactivity was localized in the outer segments of the photoreceptors (A, B), and in the ganglion cells (A, C), while a weak signal is observed in the outer plexiform layer (A, white arrows). No signal was detected in the outer segments or in ganglion cells using the blocking peptide (D, E) or in retina obtained from MT₁^−/− mice (F, G). OS = outer segments; IS = inner segments; ONL = outer nuclear layer; INL = inner nuclear layer; GCL  = ganglion cell layer. Micrographs are representative of results obtained from at least four animals for each experimental condition (Bottom panel).</p