Case Report: Longitudinal Evaluation and Treatment of a Melanoma-Associated Retinopathy Patient.

Melanoma-associated retinopathy (MAR) is a paraneoplastic syndrome associated with cutaneous metastatic melanoma in which patients develop vision deficits that include reduced night vision, poor contrast sensitivity, and photopsia. MAR is caused by autoantibodies targeting TRPM1, an ion channel found in melanocytes and retinal ON-bipolar cells (ON-BCs). The visual symptoms arise when TRPM1 autoantibodies enter ON-BCs and block the function of TRPM1, thus detection of TRPM1 autoantibodies in patient serum is a key criterion in diagnosing MAR. Electroretinograms are used to measure the impact of TRPM1 autoantibodies on ON-BC function and represent another important diagnostic tool for MAR. To date, MAR case reports have included one or both diagnostic components, but only for a single time point in the course of a patient's disease. Here, we report a case of MAR supported by longitudinal analysis of serum autoantibody detection, visual function, ocular inflammation, vascular integrity, and response to slow-release intraocular corticosteroids. Integrating these data with the patient's oncological and ophthalmological records reveals novel insights regarding MAR pathogenesis, progression, and treatment, which may inform new research and expand our collective understanding of the disease. In brief, we find TRPM1 autoantibodies can disrupt vision even when serum levels are barely detectable by western blot and immunohistochemistry; intraocular dexamethasone treatment alleviates MAR visual symptoms despite high levels of circulating TRPM1 autoantibodies, implicating antibody access to the retina as a key factor in MAR pathogenesis. Elevated inflammatory cytokine levels in the patient's eyes may be responsible for the observed damage to the blood-retinal barrier and subsequent entry of autoantibodies into the retina.

Effects of Paraquat, Dextran Sulfate Sodium, and Irradiation on Behavioral and Cognitive Performance and the Gut Microbiome in A53T and A53T-L444P Mice.

Heterozygous carriers of the glucocerebrosidase 1 (GBA) L444P Gaucher mutation have an increased risk of developing Parkinson's disease (PD). The GBA mutations result in elevated alpha synuclein (aSyn) levels. Heterozygous mice carrying one allele with the L444P mutation knocked-into the mouse gene show increased aSyn levels and are more sensitive to motor deficits following exposure to the neurotoxin (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) MPTP than wild-type mice. Paraquat (PQ), a herbicide, increases PD risk in most studies. Its effects on the brain involve alterations in the gut microbiome. Exposure to dextran sulfate sodium (DSS), a mouse model of colitis, can be used to determine whether gut microbiome alterations are sufficient to induce PD-relevant phenotypes. We rederived the A53T-L444P and A53T mouse lines to assess whether PQ, PQ in combination with radiation exposure (IR), and DSS have differential effects in A53T and A53T-L444P mice and whether these effects are associated with alterations in the gut microbiome. PQ and PQ + IR have differential effects in A53T and A53T-L444P mice. In contrast, effects of DSS are only seen in A53T-L444P mice. Exposure and genotype modulate the relationship between the gut microbiome and behavioral performance. The gut microbiome may be an important mediator of how environmental exposures or genetic mutations yield behavioral and cognitive impacts.

Trophoblast glycoprotein is required for efficient synaptic vesicle exocytosis from retinal rod bipolar cells.

Introduction: Rod bipolar cells (RBCs) faithfully transmit light-driven signals from rod photoreceptors in the outer retina to third order neurons in the inner retina. Recently, significant work has focused on the role of leucine-rich repeat (LRR) proteins in synaptic development and signal transduction at RBC synapses. We previously identified trophoblast glycoprotein (TPBG) as a novel transmembrane LRR protein localized to the dendrites and axon terminals of RBCs. Methods: We examined the effects on RBC physiology and retinal processing of TPBG genetic knockout in mice using immunofluorescence and electron microscopy, electroretinogram recording, patch-clamp electrophysiology, and time-resolved membrane capacitance measurements. Results: The scotopic electroretinogram showed a modest increase in the b-wave and a marked attenuation in oscillatory potentials in the TPBG knockout. No effect of TPBG knockout was observed on the RBC dendritic morphology, TRPM1 currents, or RBC excitability. Because scotopic oscillatory potentials primarily reflect RBC-driven rhythmic activity of the inner retina, we investigated the contribution of TPBG to downstream transmission from RBCs to third-order neurons. Using electron microscopy, we found shorter synaptic ribbons in TPBG knockout axon terminals in RBCs. Time-resolved capacitance measurements indicated that TPBG knockout reduces synaptic vesicle exocytosis and subsequent GABAergic reciprocal feedback without altering voltage-gated Ca2+ currents. Discussion: TPBG is required for normal synaptic ribbon development and efficient neurotransmitter release from RBCs to downstream cells. Our results highlight a novel synaptic role for TPBG at RBC ribbon synapses and support further examination into the mechanisms by which TPBG regulates RBC physiology and circuit function.

A sign-inverted receptive field of inhibitory interneurons provides a pathway for ON-OFF interactions in the retina.

A fundamental organizing plan of the retina is that visual information is divided into ON and OFF streams that are processed in separate layers. This functional dichotomy originates in the ON and OFF bipolar cells, which then make excitatory glutamatergic synapses onto amacrine and ganglion cells in the inner plexiform layer. We have identified an amacrine cell (AC), the sign-inverting (SI) AC, that challenges this fundamental plan. The glycinergic, ON-stratifying SI-AC has OFF light responses. In opposition to the classical wiring diagrams, it receives inhibitory inputs from glutamatergic ON bipolar cells at mGluR8 synapses, and excitatory inputs from an OFF wide-field AC at electrical synapses. This "inhibitory ON center - excitatory OFF surround" receptive-field of the SI-AC allows it to use monostratified dendrites to conduct crossover inhibition and push-pull activation to enhance light detection by ACs and RGCs in the dark and feature discrimination in the light.

Shedding light on myopia by studying complete congenital stationary night blindness.

Myopia is the most common eye disorder, caused by heterogeneous genetic and environmental factors. Rare progressive and stationary inherited retinal disorders are often associated with high myopia. Genes implicated in myopia encode proteins involved in a variety of biological processes including eye morphogenesis, extracellular matrix organization, visual perception, circadian rhythms, and retinal signaling. Differentially expressed genes (DEGs) identified in animal models mimicking myopia are helpful in suggesting candidate genes implicated in human myopia. Complete congenital stationary night blindness (cCSNB) in humans and animal models represents an ON-bipolar cell signal transmission defect and is also associated with high myopia. Thus, it represents also an interesting model to identify myopia-related genes, as well as disease mechanisms. While the origin of night blindness is molecularly well established, further research is needed to elucidate the mechanisms of myopia development in subjects with cCSNB. Using whole transcriptome analysis on three different mouse models of cCSNB (in Gpr179-/-, Lrit3-/- and Grm6-/-), we identified novel actors of the retinal signaling cascade, which are also novel candidate genes for myopia. Meta-analysis of our transcriptomic data with published transcriptomic databases and genome-wide association studies from myopia cases led us to propose new biological/cellular processes/mechanisms potentially at the origin of myopia in cCSNB subjects. The results provide a foundation to guide the development of pharmacological myopia therapies.

Light-dependent changes in the outer plexiform layer of the mouse retina.

The ability of the visual system to relay meaningful information over a wide range of lighting conditions is critical to functional vision, and relies on mechanisms of adaptation within the retina that adjust sensitivity and gain as ambient light changes. Photoreceptor synapses represent the first stage of image processing in the visual system, thus activity-driven changes at this site are a potentially powerful, yet under-studied means of adaptation. To gain insight into these mechanisms, the abundance and distribution of key synaptic proteins involved in photoreceptor to ON-bipolar cell transmission were compared between light-adapted mice and mice subjected to prolonged dark exposure (72 hours), by immunofluorescence confocal microscopy and immunoblotting. We also tested the effects on protein abundance and distribution of 0.5-4 hours of light exposure following prolonged darkness. Proteins examined included the synaptic ribbon protein, ribeye, and components of the ON-bipolar cell signal transduction pathway (mGluR6, TRPM1, RGS11, GPR179, Goα). The results indicate a reduction in immunoreactivity for ribeye, TRPM1, mGluR6, and RGS11 following prolonged dark exposure compared to the light-adapted state, but a rapid restoration of the light-adapted pattern upon light exposure. Electron microscopy revealed similar ultrastructure of light-adapted and dark-adapted photoreceptor terminals, with the exception of electron dense vesicles in dark-adapted but not light-adapted ON-bipolar cell dendrites. To assess synaptic transmission from photoreceptors to ON-bipolar cells, we recorded electroretinograms after different dark exposure times (2, 16, 24, 48, 72 hours) and measured the b-wave to a-wave ratios. Consistent with the reduction in synaptic proteins, the b/a ratios were smaller following prolonged dark exposure (48-72 hours) compared to 16 hours dark exposure (13-21%, depending on flash intensity). Overall, the results provide evidence of light/dark-dependent plasticity in photoreceptor synapses at the biochemical, morphological, and physiological levels.

Mice Lacking Gpr179 with Complete Congenital Stationary Night Blindness Are a Good Model for Myopia.

Mutations in GPR179 are one of the most common causes of autosomal recessive complete congenital stationary night blindness (cCSNB). This retinal disease is characterized in patients by impaired dim and night vision, associated with other ocular symptoms, including high myopia. cCSNB is caused by a complete loss of signal transmission from photoreceptors to ON-bipolar cells. In this study, we hypothesized that the lack of Gpr179 and the subsequent impaired ON-pathway could lead to myopic features in a mouse model of cCSNB. Using ultra performance liquid chromatography, we show that adult Gpr179-/- mice have a significant decrease in both retinal dopamine and 3,4-dihydroxyphenylacetic acid, compared to Gpr179+/+ mice. This alteration of the dopaminergic system is thought to be correlated with an increased susceptibility to lens-induced myopia but does not affect the natural refractive development. Altogether, our data added a novel myopia model, which could be used to identify therapeutic interventions.

Neuronal metabotropic glutamate receptor 8 protects against neurodegeneration in CNS inflammation.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system with continuous neuronal loss. Treatment of clinical progression remains challenging due to lack of insights into inflammation-induced neurodegenerative pathways. Here, we show that an imbalance in the neuronal receptor interactome is driving glutamate excitotoxicity in neurons of MS patients and identify the MS risk-associated metabotropic glutamate receptor 8 (GRM8) as a decisive modulator. Mechanistically, GRM8 activation counteracted neuronal cAMP accumulation, thereby directly desensitizing the inositol 1,4,5-trisphosphate receptor (IP3R). This profoundly limited glutamate-induced calcium release from the endoplasmic reticulum and subsequent cell death. Notably, we found Grm8-deficient neurons to be more prone to glutamate excitotoxicity, whereas pharmacological activation of GRM8 augmented neuroprotection in mouse and human neurons as well as in a preclinical mouse model of MS. Thus, we demonstrate that GRM8 conveys neuronal resilience to CNS inflammation and is a promising neuroprotective target with broad therapeutic implications.

Suppression of cGMP-Dependent Photoreceptor Cytotoxicity With Mycophenolate Is Neuroprotective in Murine Models of Retinitis Pigmentosa.

Purpose: To determine the effect of mycophenolate mofetil (MMF) on retinal degeneration on two mouse models of retinitis pigmentosa. Methods: Intraperitoneal injections of MMF were administered daily in rd10 and c57 mice starting at postoperative day 12 (P12) and rd1 mice starting at P8. The effect of MMF was assessed with optical coherence tomography, immunohistochemistry, electroretinography, and OptoMotry. Whole retinal cyclic guanosine monophosphate (cGMP) and mycophenolic acid levels were quantified with mass spectrometry. Photoreceptor cGMP cytotoxicity was evaluated with cell counts of cGMP immunostaining. Results: MMF treatment significantly delays the onset of retinal degeneration and cGMP-dependent photoreceptor cytotoxicity in rd10 and rd1 mice, albeit a more modest effect in the latter. In rd10 mice, treatment with MMF showed robust preservation of the photoreceptors up to P22 with associated suppression of cGMP immunostaining and microglial activation; The neuroprotective effect diminished after P22, but outer retinal thickness was still significantly thicker by P35 and OptoMotry response was significantly better up to P60. Whereas cGMP immunostaining of the photoreceptors were present in rd10 and rd1 mice, hyperphysiological whole retinal cGMP levels were observed only in rd1 mice. Conclusions: Early treatment with MMF confers potent neuroprotection in two animal models of RP by suppressing the cGMP-dependent common pathway for photoreceptor cell death. The neuroprotective effect of MMF on cGMP-dependent cytotoxicity occurs independently of the presence of hyperphysiological whole retinal cGMP levels. Thus our data suggest that MMF may be an important new class of neuroprotective agent that could be useful in the treatment of patients with RP.

Identification and characterization of novel TRPM1 autoantibodies from serum of patients with melanoma-associated retinopathy.

Melanoma-associated retinopathy (MAR) is a rare paraneoplastic retinal disorder usually occurring in the context of metastatic melanoma. Patients present with night blindness, photopsias and a constriction of the visual field. MAR is an auto-immune disorder characterized by the production of autoantibodies targeting retinal proteins, especially autoantibodies reacting to the cation channel TRPM1 produced in melanocytes and ON-bipolar cells. TRPM1 has at least three different isoforms which vary in the N-terminal region of the protein. In this study, we report the case of three new MAR patients presenting different anti-TRPM1 autoantibodies reacting to the three isoforms of TRPM1 with variable binding affinity. Two sera recognized all isoforms of TRPM1, while one recognized only the two longest isoforms upon immunolocalization studies on overexpressing cells. Similarly, the former two sera reacted with all TRPM1 isoforms on western blot, but an immunoprecipitation enrichment step was necessary to detect all isoforms with the latter serum. In contrast, all sera labelled ON-bipolar cells on Tprm1+/+ but not on Trpm1-/- mouse retina as shown by co-immunolocalization. This confirms that the MAR sera specifically detect TRPM1. Most likely, the anti-TRPM1 autoantibodies of different patients vary in affinity and concentration. In addition, the binding of autoantibodies to TRPM1 may be conformation-dependent, with epitopes being inaccessible in some constructs (truncated polypeptides versus full-length TRPM1) or applications (western blotting versus immunohistochemistry). Therefore, we propose that a combination of different methods should be used to test for the presence of anti-TRPM1 autoantibodies in the sera of MAR patients.

TRPM1 Autoantibodies in Melanoma Patients Without Self-Reported Visual Symptoms.

Purpose: Melanoma-associated retinopathy (MAR) is a paraneoplastic syndrome associated with cutaneous malignant melanoma (CMM). Visual symptoms include night blindness, photopsia, and reduced-contrast sensitivity. An abnormal ERG b-wave and the presence of anti-bipolar cell autoantibodies, including autoantibodies reacting with the ON-bipolar cell TRPM1 channel, help to confirm the diagnosis. The goal of this study was to determine if CMM patients without visual symptoms also express anti-TRPM1 autoantibodies. Methods: Serum samples from 15 CMM patients were tested using three assays: immunofluorescent labeling of TRPM1-transfected HEK cells, immunofluorescent labeling of retinal sections from wild-type and TRPM1 knockout mice, and immunoblot detection of a bacterially produced recombinant TRPM1 peptide. Results: Serum specimens from 5 of the 15 CMM patients without declared visual symptoms were positive for anti-TRPM1 autoantibodies in at least one of the three assays. One of 50 control sera from patients not known to have cancer was also weakly reactive with the TRPM1 peptide. Conclusions: Autoantibodies against TRPM1 are present in CMM patient sera without self-reported visual symptoms. Most patients had advanced (stage III and IV) disease and were undergoing aggressive treatments, including immunotherapy. It is unknown if immunotherapy affects the expression of TRPM1 autoantibodies. The presence of TRPM1 autoantibodies may predispose patients for MAR.

Effects of Sub-Chronic MPTP Exposure on Behavioral and Cognitive Performance and the Microbiome of Wild-Type and mGlu8 Knockout Female and Male Mice.

Motor dysfunction is a hallmark of Parkinson's disease (PD); however, non-motor symptoms such as gastrointestinal dysfunction often arise prior to motor symptoms. Alterations in the gut microbiome have been proposed as the earliest event in PD pathogenesis. PD symptoms often demonstrate sex differences. Glutamatergic neurotransmission has long been linked to PD pathology. Metabotropic glutamate receptors (mGlu), a family of G protein-coupled receptors, are divided into three groups, with group III mGlu receptors mainly localized presynaptically where they can inhibit glutamate release in the CNS as well as in the gut. Additionally, the gut microbiome can communicate with the CNS via the gut-brain axis. Here, we assessed whether deficiency of metabotropic glutamate receptor 8 (mGlu8), group III mGlu, modulates the effects of the neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), on behavioral and cognitive performance in female and male mice. We studied whether these effects are associated with changes in striatal tyrosine hydroxylase (TH) levels and the gut microbiome. Two-week sub-chronic MPTP increased activity of female and male wild-type (WT) and mGlu8 knockout (KO) mice in the open field. MPTP also showed genotype- and sex-dependent effects. MPTP increased the time WT, but not KO, females and males spent exploring objects. In WT mice, MPTP improved sensorimotor function in males but impaired it in females. Further, MPTP impaired cued fear memory in WT, but not KO, male mice. MPTP reduced striatal TH levels in WT and KO mice but these effects were only pronounced in males. MPTP treatment and genotype affected the diversity of the gut microbiome. In addition, there were significant associations between microbiome α-diversity and sensorimotor performance, as well as microbiome composition and fear learning. These results indicate that specific taxa may directly affect motor and fear learning or that the same physiological effects that enhance both forms of learning also alter diversity of the gut microbiome. MPTP's effect on motor and cognitive performance may then be, at least in part, be mediated by the gut microbiome. These data also support mGlu8 as a novel therapeutic target for PD and highlight the importance of including both sexes in preclinical studies.

The future of graduate and postdoctoral training in the biosciences.

This article summarizes the outcomes of the second national conference on the Future of Bioscience Graduate and Postdoctoral Training. Five topics were addressed during the conference: diversity in leadership positions; mentoring; modernizing the curriculum; experiential learning; and the need for better data on trainees. The goal of the conference was to develop a consensus around these five topics and to recommend policies that can be implemented by academic and research institutions and federal funding agencies in the United States.

Autoantibodies in Melanoma-Associated Retinopathy Recognize an Epitope Conserved Between TRPM1 and TRPM3.

Purpose: Melanoma-associated retinopathy (MAR) is a paraneoplastic syndrome associated with malignant melanoma and the presence of anti-retinal autoantibodies, including autoantibodies against transient receptor potential melanopsin 1 (TRPM1), a cation channel expressed by both melanocytes and retinal bipolar cells. The goal of this study was to further map the antigenic epitope. Methods: Patient sera were tested by immunofluorescence and Western blotting on HEK293 cells transfected with enhanced green fluorescent protein (EGFP)-TRPM1 fusion constructs and mouse retina sections. Results: The epitope recognized by MAR patient sera was mapped to a region encoded by exons 9 and 10 of the human TRPM1 gene. This region of TRPM1 is highly conserved with TRPM3, and indeed MAR sera were found to cross-react with TRPM3, a closely related channel expressed in the retinal pigment epithelium (RPE). Conclusions: These results indicate that TRPM1 autoantibodies in MAR patient sera recognize a short, intracellular segment of TRPM1. Cross-reactivity with TRPM3 in the RPE may account for other visual symptoms that are experienced by some MAR patients such as retinal and RPE detachments. We propose that TRPM1 autoantibodies are generated in response to abnormal TRPM1 polypeptides encoded by an alternate mRNA splice variant expressed by malignant melanocytes.

The Effect of PKCα on the Light Response of Rod Bipolar Cells in the Mouse Retina.

PURPOSE: Protein kinase C α (PKCα) is abundantly expressed in rod bipolar cells (RBCs) in the retina, yet the physiological function of PKCα in these cells is not well understood. To elucidate the role of PKCα in visual processing in the eye, we examined the effect of genetic deletion of PKCα on the ERG and on RBC light responses in the mouse. METHODS: Immunofluorescent labeling was performed on wild-type (WT), TRPM1 knockout, and PKCα knockout (PKC-KO) retina. Scotopic and photopic ERGs were recorded from WT and PKC-KO mice. Light responses of RBCs were measured using whole-cell recordings in retinal slices from WT and PKC-KO mice. RESULTS: Protein kinase C alpha expression in RBCs is correlated with the activity state of the cell. Rod bipolar cells dendrites are a major site of PKCα phosphorylation. Electroretinogram recordings indicated that loss of PKCα affects the scotopic b-wave, including a larger peak amplitude, longer implicit time, and broader width of the b-wave. There were no differences in the ERG a- or c-wave between PKCα KO and WT mice, indicating no measurable effect of PKCα in photoreceptors or the RPE. The photopic ERG was unaffected consistent with the lack of detectable PKCα in cone bipolar cells. Whole-cell recordings from RBCs in PKC-KO retinal slices revealed that, compared with WT, RBC light responses in the PKC-KO retina are delayed and of longer duration. CONCLUSIONS: Protein kinase C alpha plays an important modulatory role in RBCs, regulating both the peak amplitude and temporal properties of the RBC light response in the rod visual pathway.

LRIT3 is essential to localize TRPM1 to the dendritic tips of depolarizing bipolar cells and may play a role in cone synapse formation.

Mutations in LRIT3 lead to complete congenital stationary night blindness (cCSNB). The exact role of LRIT3 in ON-bipolar cell signaling cascade remains to be elucidated. Recently, we have characterized a novel mouse model lacking Lrit3 [no b-wave 6, (Lrit3(nob6/nob6) )], which displays similar abnormalities to patients with cCSNB with LRIT3 mutations. Here we compare the localization of components of the ON-bipolar cell signaling cascade in wild-type and Lrit3(nob6/nob6) retinal sections by immunofluorescence confocal microscopy. An anti-LRIT3 antibody was generated. Immunofluorescent staining of LRIT3 in wild-type mice revealed a specific punctate labeling in the outer plexiform layer (OPL), which was absent in Lrit3(nob6/nob6) mice. LRIT3 did not co-localize with ribeye or calbindin but co-localized with mGluR6. TRPM1 staining was severely decreased at the dendritic tips of all depolarizing bipolar cells in Lrit3(nob6/nob6) mice. mGluR6, GPR179, RGS7, RGS11 and Gß5 immunofluorescence was absent at the dendritic tips of cone ON-bipolar cells in Lrit3(nob6/nob6) mice, while it was present at the dendritic tips of rod bipolar cells. Furthermore, peanut agglutinin (PNA) labeling was severely reduced in the OPL in Lrit3(nob6/nob6) mice. This study confirmed the localization of LRIT3 at the dendritic tips of depolarizing bipolar cells in mouse retina and demonstrated the dependence of TRPM1 localization on the presence of LRIT3. As tested components of the ON-bipolar cell signaling cascade and PNA revealed disrupted localization, an additional function of LRIT3 in cone synapse formation is suggested. These results point to a possibly different regulation of the mGluR6 signaling cascade between rod and cone ON-bipolar cells.

Voriconazole, an antifungal triazol that causes visual side effects, is an inhibitor of TRPM1 and TRPM3 channels.

PURPOSE: Administration of voriconazole, an antifungal triazole, causes transient visual disturbances in patients and attenuates the b-wave of the ERG. We sought to identify the retinal target of voriconazole underlying the effect on the ERG b-wave. METHODS: Electroretinograms were recorded from mice before and after intraperitoneal injection of voriconazole. The effect of voriconazole on ON-bipolar cells was tested by patch-clamp recordings of ON-bipolar cells in mouse retinal slices. Effects of voriconazole on mGluR6 and TRPM3 were assessed by patch-clamp recordings of Chinese hamster ovary (CHO) and HEK293 cells transfected with either TRPM3 or mGluR6 plus Kir3.1/Kir3.4. RESULTS: Voriconazole attenuated the ERG b-wave in mice, and inhibited ON-bipolar cell responses evoked by application of CPPG, an mGluR6 antagonist, onto the ON-bipolar cell dendrites, indicating that voriconazole blocks a step in the mGluR6-TRPM1 signal transduction pathway. Voriconazole almost completely blocked capsaicin-activated currents in ON-bipolar cells, which have been attributed to direct activation of the TRPM1 cation channel. Furthermore, application of voriconazole to CHO cells expressing TRPM3, a closely related channel to TRPM1, showed that voriconazole reversibly blocked pregnenolone sulfate-stimulated TRPM3 currents in transfected cells. In contrast, voriconazole only slightly inhibited mGluR6-mediated activation of G-protein activated inward rectifier potassium (GIRK) currents in cotransfected cells, suggesting that mGluR6 is not the primary target of voriconazole in ON-bipolar cells. CONCLUSIONS: The visual disturbances associated with voriconazole are likely due to block of TRPM1 channels in retinal ON-bipolar cells. Other neurological effects of voriconazole may be due to block of TRPM3 channels expressed in the brain.

TRPM3 expression in mouse retina.

Transient receptor potential (TRP) channels constitute a large family of cation permeable ion channels that serve crucial functions in sensory systems by transducing environmental changes into cellular voltage and calcium signals. Within the retina, two closely related members of the melastatin TRP family, TRPM1 and TRPM3, are highly expressed. TRPM1 has been shown to be required for the depolarizing response to light of ON-bipolar cells, but the role of TRPM3 in the retina is unknown. Immunohistochemical staining of mouse retina with an antibody directed against the C-terminus of TRPM3 labeled the inner plexiform layer (IPL) and a subset of cells in the ganglion cell layer. Within the IPL, TRPM3 immunofluorescence was markedly stronger in the OFF sublamina than in the ON sublamina. Electroretinogram recordings showed that the scotopic and photopic a- and b-waves of TRPM3(-/-) mice are normal indicating that TRPM3 does not play a major role in visual processing in the outer retina. TRPM3 activity was measured by calcium imaging and patch-clamp recording of immunopurified retinal ganglion cells. Application of the TRPM3 agonist, pregnenolone sulfate (PS), stimulated increases in intracellular calcium in ~40% of cells from wild type and TRPM1(­/­) mice, and the PS-stimulated increases in calcium were blocked by co-application of mefenamic acid, a TRPM3 antagonist. No PS-stimulated changes in fluorescence were observed in ganglion cells from TRPM3(-/-) mice. Similarly, PS-stimulated currents that could be blocked by mefenamic acid were recorded from wild type retinal ganglion cells but were absent in ganglion cells from TRPM3-/- mice.

Novel metabotropic glutamate receptor 4 and glutamate receptor 8 therapeutics for the treatment of anxiety.

INTRODUCTION: The fast actions of the excitatory neurotransmitter glutamate are mediated by glutamate-gated ion channels (ionotropic Glu receptors). Metabotropic glutamate receptors (mGlus) are coupled to second messenger pathways via G proteins and modulate glutamatergic and GABAergic neurotransmission. Of the eight different types of mGlus (mGlu1-mGlu8), mGlu4, mGlu6, mGlu7 and mGlu8 are members of group III. Except for mGlu6, group III receptors are generally located presynaptically and regulate neurotransmitter release. Because of their role in modulating excitatory neurotransmission, mGlus are attractive targets for therapies aimed at treating anxiety disorders. AREAS COVERED: In this review, the authors discuss the role of mGlu4 and mGlu8 in anxiety disorders. They also discuss how mGlu4 and mGlu8 have distinct expression patterns in the brain, which might have related functions. Finally, the authors discuss how compounds that target more than one mGlu receptor might be therapeutically more effective. EXPERT OPINION: mGlu4 might compensate for mGlu8 deficiency, and deficiency of both receptors might result in a more pronounced phenotype than deficiency of either receptor alone. The distinct and overlapping anatomical distribution and functions of mGlu4 and mGlu8 suggest that both receptors, either individually or combined, are attractive therapeutic targets in anxiety disorders, post-traumatic stress disorder, Parkinson's disease, and multiple sclerosis.

Characterization of the novel positive allosteric modulator of the metabotropic glutamate receptor 4 ADX88178 in rodent models of neuropsychiatric disorders.

There is growing evidence that activation of metabotropic glutamate receptor 4 (mGlu4) leads to anxiolytic- and antipsychotic-like efficacy in rodent models, yet its relevance to depression-like reactivity remains unclear. Here, we present the pharmacological evaluation of ADX88178 [5-methyl-N-(4-methylpyrimidin-2-yl)-4-(1H-pyrazol-4-yl)thiazol-2-amine], a novel potent, selective, and brain-penetrant positive allosteric modulator of the mGlu4 receptor in rodent models of anxiety, obsessive compulsive disorder (OCD), fear, depression, and psychosis. ADX88178 dose-dependently reduced the number of buried marbles in the marble burying test and increased open-arm exploration in the elevated plus maze (EPM) test, indicative of anxiolytic-like efficacy. Target specificity of the effect in the EPM test was confirmed using male and female mGlu4 receptor knockout mice. In mice, ADX88178 reduced the likelihood of conditioned freezing in the acquisition phase of the fear conditioning test, yet had no carryover effect in the expression phase. Also, ADX88178 dose-dependently reduced duration of immobility in the forced swim test, indicative of antidepressant-like efficacy. ADX88178 reduced DOI (2,5-dimethoxy-4-iodoamphetamine)-mediated head twitches (albeit with no dose-dependency), and MK-801 [(5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine]-induced locomotor hyperactivity in mice, but was inactive in the conditioned avoidance response test in rats. The compound showed good specificity as it had no effect on locomotor activity in mice and rats at efficacious doses. Thus, allosteric activation of mGlu4 receptors can be a promising new therapeutic approach for treatment of anxiety, OCD, fear-related disorders, and psychosis.