Mechanisms of calcium-dependent neurotransmission in photoreceptors

Rod and cone photoreceptors initiate vision by transforming light into graded membrane voltage changes that in turn dictate the rate of continuous Ca2+-dependent neurotransmission to postsynaptic neurons. Continuous release relies on synaptic ribbons at photoreceptor active zones, which organize exocytotic proteins and deliver vesicles to release sites near voltage-gated Ca2+ channels. Individual cones possess multiple ribbon synapses at which they contact postsynaptic neurons. We examined heterogeneity in signaling at individual ribbon synapses in salamander cones by measuring the voltage dependence of Ca2+ currents (ICa) and Ca2+ influx at individual ribbon release sites. Ca2+ signals at individual ribbons varied more in amplitude and voltage of half-maximal activation (V50) than whole-cell ICa, suggesting that Ca2+ signals differ significantly between individual ribbons within cones. The ability of ribbons to function independently was further demonstrated by showing that inhibitory feedback from individual horizontal cells (HCs) affected Ca2+ dynamics at certain ribbons but not others within a single cone. Individual ribbon synapses operating independently from one another broadens the range of transformations available to cones in the transmission of light responses to downstream neurons. Rods and cones release vesicles with higher sensitivity and a shallower exponential relationship to presynaptic Ca2+ than most neurons. These characteristics are typically attributed to properties of the vesicular Ca2+ sensor that triggers release. By conditionally deleting Synaptotagmin 1 (Syt1) from rods and cones, we show that Syt1 is the chief Ca2+ sensor that operates in mouse photoreceptors. Removal of Syt1 reduced b-waves but not a-waves of the electroretinogram (ERG) and diminished fast exocytosis evoked by brief depolarizing steps as evaluated by single-cell recordings. Slower and spontaneous forms of release were not eliminated, suggesting that other Ca2+ sensors are also present. Synaptic anatomy was unaltered in Syt1 mutant mice, suggesting that synapse development and maintenance occur in a Syt1-independent manner. Our results indicate that Syt1 is essential for the transmission of light responses across photoreceptor synapses and suggest that the Ca2+ dependence of release can be shaped by factors other than the intrinsic properties of the Ca2+ sensor

Retinal synaptic function in the absence of the on pathway.

Complete Congenital Stationary Night Blindness (cCSNB) is a rare hereditary retinal disorder characterized by abnormal night vision. cCSNB is caused by postsynaptic defects in On bipolar cells (BCs) and is identified by the presence of an electroretinogram (ERG) with a normal a-wave, corresponding to photoreceptor function, and the absence of a b-wave, corresponding to a failure of On BC function. Through the study of genetic mutations in mouse that result in no b-wave ERG phenotypes, several proteins have been identified that play crucial roles in On BC signal transmission. I focused on four mouse models of cCSNB; Nyxnob (Nyctalopin mutant), mGluR6-/- (mGluR6 knockout), Gpr179nob5 (GPR179 mutant), and Lrit3-/- (LRIT3 knockout). These mutations effect proteins expressed by On BCs (rod and On Cone BCs). While all models of cCSNB share a no b-wave ERG phenotype I have discovered that several models differ. The differences between cCSNB animal models provide important clues into the functional roles of the proteins effected by the mutations. Specifically, Nyxnob retinal ganglion cells (RGCs), the output neurons of the retina, exhibit robust 3-5 Hz rhythmic spiking while mGluR6-/- RGCs rarely do. I explored potential mechanisms which underlie this phenomenon, not only by examining RGC activity, but also the properties of the upstream rod BCs which provide excitatory input to RGCs. I found that differences in the resting state of Nyxnob and mGluR6-/- rod BCs correlate with the differences in RGC rhythmic spiking activity. Also, I discovered that nyctalopin is required for normal potassium conductance in rod BCs. Additionally, I examined the role of two recently identified proteins expressed in On BCs, GPR179 (Peachey et al., 2012; Ray et al., 2014) and LRIT3 (Zeitz et al., 2013; Neuille et al., 2014). I discovered that GPR179 sets the sensitivity of the TRPM1 channel and is critical for a normal light-evoked response in rod BCs. I also discovered that LRIT3 is critical for the modulation and expression of TRPM1 channels in rod BC dendritic tips. My data not only add to the literature on animal models of cCSNB, but to the understanding of retinal circuitry in the normal retina

Membrane properties of cones and ganglion cells of the salamander retina

The aim of this thesis was to investigate the membrane properties of cone photoreceptors and ganglion cells of the salamander retina and to determine their role in the processing of the visual signal. Experimental investigations were carried out on cells in the intact retina and also in cells that had been isolated from the retina by enzymatic dissociation. Glutamate is thought to be the neurotransmitter released from vertebrate photoreceptors. Glutamate gates channels in postsynaptic bipolar and horizontal cells, but there have been no exhaustive studies of the effects of glutamate on the photoreceptors themselves. In patch-clamp recordings from both isolated cones and cones in the intact salamander retina, glutamate was found to activate a current carried largely by chloride ions, which is localized to the synaptic terminal of the cone. This suggests that glutamate released from a cone terminal may act on "autoreceptors" on that terminal, modulating its own release. This may be important as a mechanism for increasing the gain of cone phototransduction. The membrane properties of ganglion cells determine how visual information is coded for transmission to the brain. Ganglion cells have previously been shown to exist as at least two types, sustained and transient, in terms of the pattern of action potentials produced in response to illumination. The origin of transience in ganglion cells in unclear. Salamander ganglion cells show sustained or transient responses to the injection of current mimicking light-induced synaptic input. Using the whole-cell recording method, the properties of both voltage-gated currents and excitatory and inhibitory neurotransmitter-gated currents were investigated in voltage-clamped salamander ganglion cells. On the basis of these results, it is suggested that transience in the response of ganglion cells may in part be due to the properties of the voltage-gated membrane currents present in these cells

High-Pass Filtering of Input Signals by the Ih Current in a Non-Spiking Neuron, the Retinal Rod Bipolar Cell

Hyperpolarization–activated cyclic nucleotide–sensitive (HCN) channels mediate the If current in heart and Ih throughout the nervous system. In spiking neurons Ih participates primarily in different forms of rhythmic activity. Little is known, however, about its role in neurons operating with graded potentials as in the retina, where all four channel isoforms are expressed. Intriguing evidence for an involvement of Ih in early visual processing are the side effects reported, in dim light or darkness, by cardiac patients treated with HCN inhibitors. Moreover, electroretinographic recordings indicate that these drugs affect temporal processing in the outer retina. Here we analyzed the functional role of HCN channels in rod bipolar cells (RBCs) of the mouse. Perforated–patch recordings in the dark–adapted slice found that RBCs exhibit Ih, and that this is sensitive to the specific blocker ZD7288. RBC input impedance, explored by sinusoidal frequency–modulated current stimuli (0.1–30 Hz), displays band–pass behavior in the range of Ih activation. Theoretical modeling and pharmacological blockade demonstrate that high–pass filtering of input signals by Ih, in combination with low–pass filtering by passive properties, fully accounts for this frequency–tuning. Correcting for the depolarization introduced by shunting through the pipette–membrane seal, leads to predict that in darkness Ih is tonically active in RBCs and quickens their responses to dim light stimuli. Immunohistochemistry targeting candidate subunit isoforms HCN1–2, in combination with markers of RBCs (PKC) and rod–RBC synaptic contacts (bassoon, mGluR6, Kv1.3), suggests that RBCs express HCN2 on the tip of their dendrites. The functional properties conferred by Ih onto RBCs may contribute to shape the retina's light response and explain the visual side effects of HCN inhibitors

A Presynaptic Role for Nitric Oxide at a GABAergic Synapse

Amacrine cells are a class of retinal interneurons that process the visual signal in the inner retina. Several subtypes of amacrine cells express nitric oxide synthase and produce nitric oxide (NO), making NO a possible regulator of amacrine cell function. My dissertation research tests the hypothesis that NO alters amacrine cell GABAergic synaptic output. To investigate this, I made whole-cell voltage clamp recordings of cultured chick amacrine cells receiving synaptic input from other amacrine cells and Ca2+ imaging of amacrine cell dendrites, which can be presynaptic. I find that NO-dependent increases in GABAergic spontaneous postsynaptic current (sPSC) frequency are independent of soluble guanylate cyclase and action potentials. Removal of extracellular Ca2+ and buffering of cytosolic Ca2+ both inhibit the response to NO. In Ca2+ imaging experiments, I confirm that NO increases dendritic Ca2+ by activating a Ca2+ influx pathway. Neither NO-dependent dendritic Ca2+ elevation nor increase in sPSC frequency are dependent upon Ca2+ release from stores. NO also enhances evoked GABAergic responses, and because voltage-gated Ca2+ channel function is not altered by NO, the enhanced evoked release is likely due to the combination of voltage-dependent Ca2+ influx and the voltage-independent, NO-dependent Ca2+ influx. Insight into the identity of the Ca2+ channel involved in the NO response was provided by characteristics unique to the transient receptor potential canonical (TRPC) channel subunits 4 and 5: the NO-dependent increase in sPSC frequency was dependent on downstream activity of PLC, blocked by 2 mM La3+ and enhanced by 10 µM La3+. The TRPC inhibitor ML204, which preferentially blocks TRPC4, had no effect on the NO response at 10 µM, but 20 µM ML204 blocked the NO response. The TRPC inhibitor clemizole, which preferentially blocks TRPC5, blocked NO-dependent dendritic Ca2+ elevations and the increase in sPSC frequency. Genetic knockdown of TRPC5 in cultured amacrine cells using the CRISPR/Cas9 system confirms that TRPC5 mediates NO-dependent dendritic Ca2+ elevations and the increase in sPSC frequency. These results suggest that NO-dependent activation of TRPC5 at amacrine cell presynaptic sites will enhance vesicular GABA release and increase inhibition onto postsynaptic cells

Spatiotemporal response of the photoreceptor network

The retina is a specialized part of the central nervous system adapted to encoding images into electrical signals. Images are formed on the back of the eye by the lens and cornea, and photons that make up those images are absorbed by light sensitive pigments in the photoreceptors. Photon absorptions by these pigments generate a current, the photocurrent, which is modified by voltage-gated ion channels and electrical connections to adjacent photoreceptors. A voltage change in the photoreceptor is transformed into a chemical signal to downstream cells by its modulatory effect on the calcium concentration at the synapse. This thesis examines two important elements in photoreceptor function other than the photocurrent: the Ih current and electrical coupling between rods. Here, using the tiger salamander (Ambystoma tigrinum) as a model, we investigate the kinetic properties of the HCN channels responsible for the Ih current in photoreceptors, and show that they are similar in rods and cones, which in turn are similar to the known properties of the HCN1 isoform. With western blot and immunostaining, we show that the HCN1 isoform is present in retina. We also demonstrate how HCN channels modify the kinetics of the rod and cone light response to make it faster. This thesis integrates this and other data from photoreceptor ion channels into physiology-based models of rod and cone photoreceptors. Through simulation, the model of the rod demonstrates that conductance changes from the h and Kx currents largely cancel one another during the rod light response. The cone model is used to demonstrate the feasibility of two proposed mechanisms for horizontal cell to cone negative feedback. Finally, this work presents measurements of electrical coupling between rod photoreceptors in the salamander retina using both light and electrical stimuli. Using measured parameters for the coupling resistance, a model of the electrically coupled network of rod photoreceptors is developed. We use this model to demonstrate how rod-rod coupling decreases noise at the expense of attenuating sharp contrasts in visual scenes. The model predicts the tradeoff between these two factors results in an overall improvement in the signal-to-noise ratio for most perceptible stimuli. Results suggest that photoreceptor coupling is especially helpful in the perception of images with statistical qualities similar to natural scenes

Molecular and Cellular Mechanisms Underlying Somatostatin-Based Signaling in Two Model Neural Networks, the Retina and the Hippocampus

Neural inhibition plays a key role in determining the specific computational tasks of different brain circuitries. This functional \u201cbraking\u201d activity is provided by inhibitory interneurons that use different neurochemicals for signaling. One of these substances, somatostatin, is found in several neural networks, raising questions about the significance of its widespread occurrence and usage. Here, we address this issue by analyzing the somatostatinergic system in two regions of the central nervous system, the retina and the hippocampus. By comparing the available information on these structures, we have identified common motifs in the action of somatostatin that may explain its involvement in such diverse circuitries. The emerging concept is that somatostatin-based signaling, through conserved molecular and cellular mechanisms, allows neural networks to operate correctly

Roles of ON cone bipolar cell subtypes in temporal coding in the mouse retina

In the visual system, diverse image processing starts with bipolar cells, which are the second-order neurons of the retina. Thirteen subtypes of bipolar cells have been identified, which are thought to encode different features of image signaling and to initiate distinct signal-processing streams. Although morphologically identified, the functional roles of each bipolar cell subtype in visual signal encoding are not fully understood. Here, we investigated how ON cone bipolar cells of the mouse retina encode diverse temporal image signaling. We recorded bipolar cell voltage changes in response to two different input functions: sinusoidal light and step light stimuli. Temporal tuning in ON cone bipolar cells was diverse and occurred in a subtype-dependent manner. Subtypes 5s and 8 exhibited low-pass filtering property in response to a sinusoidal light stimulus, and responded with sustained fashion to step-light stimulation. Conversely, subtypes 5f, 6, 7, and XBC exhibited bandpass filtering property in response to sinusoidal light stimuli, and responded transiently to step-light stimuli. In particular, subtypes 7 and XBC were high-temporal tuning cells. We recorded responses in different ways to further examine the underlying mechanisms of temporal tuning. Current injection evoked low-pass filtering, whereas light responses in voltage-clamp mode produced bandpass filtering in all ON bipolar cells. These findings suggest that cone photoreceptor inputs shape bandpass filtering in bipolar cells, whereas intrinsic properties of bipolar cells shape low-pass filtering. Together, our results demonstrate that ON bipolar cells encode diverse temporal image signaling in a subtype-dependent manner to initiate temporal visual information-processing pathways

Multiquantal Glutamate Release from Rod Photoreceptors

Neurons communicate via Ca2+-dependent release of neurotransmitters packaged into vesicles (quanta). Some CNS neurons, especially sensory synapses, can release multiple vesicles at a time, increasing information transmission and overcoming the unreliability of a stochastic process. Ribbon-bearing neurons, including retinal photoreceptors, face the challenge of encoding sensory receptor potentials into an ever-changing train of vesicle release events. We studied release of glutamate using voltage clamp to measure anion currents activated during glutamate reuptake into presynaptic terminals (IA(glu)) of salamander and mouse rods, finding that each employ distinct mechanisms for multiquantal release. In amphibian rods, we found that 1/3 of the spontaneous IA(glu) fusion events involve synchronous fusion of multiple vesicles. By varying intracellular buffering to localize Ca2+-dependent events, we found that multiquantal release occurs near Ca2+ sources. In photoreceptors, Ca2+ influx occurs just below synaptic ribbons. Vesicles house SNARE machinery so we hypothesized that vesicles on the ribbon undergo homotypic fusion prior to exocytosis. Destruction of ribbons and disruption of the SNARE-protein syntaxin3B prevented spontaneous multiquantal release, suggesting that salamander rods are capable of multivesicular release due to homotypic fusion of vesicles along ribbons. In mouse rods, spontaneous release at −70 mV involved the stochastic fusion of single vesicles. With depolarization, glutamate release increased linearly with voltage-gated Ca2+ currents. As the membrane approached the resting potential in darkness of −40 mV, rods began to release glutamate in multivesicular bursts of 17±7 vesicles every 2801±598 ms. Release evoked by brief depolarizations and bursts both involved the same pool of ribbon-associated vesicles with fusion regulated by the vesicular Ca2+ sensor synaptotagmin-1. A second, slower component of release controlled by synaptotagmin-7 is also present in rods but not cones. We hypothesized a v role for coordinated bursts of release in transmitting single photon signals. The rate of bursting was responsive to small voltage changes of 1.0-3.5 mV and the voltage waveform that triggered bursts most effectively was similar to single photon responses. We propose that multiquantal bursts contribute to mechanisms that filter out small noisy events to improve reliable detection of single photons by the retina