ON bipolar cells are critical for the function of the ON pathway in the visual system. They express a metabotropic glutamate receptor (mGluR6) that, when activated, couples to the G o class of G protein. The channel that is primarily responsible for the synaptic response has been recently identified as the transient receptor potential cation channel subfamily M member 1 (TRPM1); TRPM1 is negatively coupled to the mGluR6/Go cascade such that activation of the cascade results in closure of the channel. Light indirectly opens TRPM1 by reducing transmitter release from presynaptic photoreceptors, resulting in a decrease in mGluR6 activation. Conversely, in the dark, binding of synaptic glutamate to mGluR6 inhibits TRPM1 current. Closure of TRPM1 by G-protein activation in the dark is a critical step in the process of ON bipolar cell signal transduction, but the precise pathway linking these two events is not understood. To address this question, we measured TRPM1 activity in retinal bipolar cells, in human ependymal melanocytes (HEMs) that endogenously express TRPM1, and in HEK293 cells transfected with TRPM1. Dialysis of the Gβγ subunit dimer, but not Gα o , closed TRPM1 channels in every cell type that we tested. In addition, activation of an endogenous G-protein-coupled receptor pathway in HEK293 cells that releases Gβγ without activating Go protein also closed TRPM1 channels. These results suggest a model in which the Gβγ dimer that is released as a result of the dissociation from Gα o upon activation of mGluR6 closes the TRPM1 channel, perhaps via a direct interaction. patch clamp | calcium imaging R etinal ON bipolar cells are connected to photoreceptors through a sign-inverting synapse. At this synapse, glutamate binds to the metabotropic glutamate receptor mGluR6, which couples to the closure of a cation-selective transduction channel. Insight into the molecular identity of the transduction channel was gained from the observation that low levels of mRNA encoding the transient receptor potential ion channel TRPM1 was associated with a form of congenital stationary night blindness (CSNB) in a population of Appaloosa horses (1). Soon after, it was shown that ON bipolar cell function was absent or severely impaired in a mouse model that lacked TRPM1 expression (2-4), although there is evidence that one or more classes of cone-driven ON bipolar cells may use an additional channel (3). Mutations in TRPM1 have now been positively linked to CSNB in humans as well (5-7). Taken together, studies of mouse, horse, and human provide convincing evidence that the ON bipolar cell transduction channel is composed of TRPM1, either alone or in conjunction with other channels. However, all these studies do not provide insight into the mechanism by which activation of mGluR6 leads to closure of TRPM1.The metabotropic receptor mGluR6 preferentially couples to the G o class of G protein, both in bipolar cells (8, 9) and expression systems (10, 11). Activation of mGluR6 and, correspondingly, Go, results in closure of TRPM1. However...

In the retina, light onset hyperpolarizes photoreceptors and depolarizes ON bipolar cells at the sign inverting photoreceptor – ON bipolar cell synapse. Transmission at this synapse is mediated by a signaling cascade comprised of the metabotropic glutamate receptor mGluR6, a G-protein containing Gαo, and the cation channel TrpM1. This system is thought to be common to both the rod and ON-cone driven pathways, which control vision under scotopic and photopic conditions, respectively. In this study, we present evidence that the rod pathway is uniquely susceptible to modulation by PKCα at the rod- rod bipolar cell synapse. Decreased production of DAG (an activator of PKC) by inhibition of PIP2 hydrolysis caused depression of the TrpM1 current. Conversely, addition of a DAG analog, OAG, potentiated the current in rod bipolar cells but not in ON-cone bipolar cells. The potentiating effects of OAG were absent both in mutant mice that lack PKCα expression and in wild type mice in which enzymatic activity of PKCα was pharmacologically inhibited. In addition, we found that, like other members of the TrpM subfamily, TrpM1 current is susceptible to voltage independent inhibition by intracellular magnesium, and that modulation by PKCα relieves this inhibition, as the potentiating effects of OAG are absent in low intracellular magnesium. We conclude that activation of PKCα initiates a modulatory mechanism at the rod – rod bipolar cell synapse whose function is to reduce inhibition of the TrpM1 current by magnesium, thereby increasing the gain of transmission at this synapse.

Service members (SMs) suffer from repeated exposure to concussions or mild TBI, which can disrupt cerebral blood flow. This effect is seen in the acute phase of TBI, but the development of vascular wall abnormalities, which can lead to luminal narrowing and abnormally elevated cerebral blood flow velocities (CBFV), has not been studied in the post concussive phase. The purpose of this study was to evaluate the transcranial Doppler (TCD) determined incidence of abnormal cerebral hemodynamics in patients with long-term effects of mTBI. Material and Methods: TCD recordings of mean CBFV in cm/sec and Pulsatility Indices (PI) were recorded from 431 SMs (9 females; ages 24-54, mean age 39.2 ± 6.4 years) who were admitted to the National Intrepid Center of Excellence 4-week Interdisciplinary Outpatient Program. TCD data were analyzed to determine whether SMs exhibited abnormal CBFV s in the middle cerebral artery, internal carotid artery (ICA), and basilar artery. All subjects consented to protocol #362504 prior to any data collection or analysis. Two standard deviation from the mean CBFV in any direction labeled as an abnormal and PI was assumed normal with values between 0.7 and 0.9 Patients with one or more vessels with abnormal CBFV’s and PI’s were labeled as abnormal. Results: Average time since last TBI was 5.1 ± 3.4 years. We observed abnormal CBFV values in 33% of SMs. 149 (35%) and 48 (11%) demonstrated abnormally high CBFV and PIs in one or more vessels, respectively. Abnormal CBFV was observed most frequently in the ICA (48%). Majority of studied patients had patchy pattern of abnormal data presented in one or few vessels. The presence of abnormal cerebral hemodynamics was inversely related to the time since the last mTBI exposure: the likelihood of abnormal CBFV values deceased with increasing time since last head injury was sustained but was still present. Conclusions: Our data reveal that a large proportion of SMs have elevated CBFVs following mTBI exposure in many years after last mTBI. We believe that head injuries due to combat and training blast and blunt force trauma exposure cause early atherosclerotic or fibrotic changes in the large vessel walls of the Circle of Willis, which results in vascular lumen narrowing and increased CBFV’s.