Electrophysiological investigations on the role of retinal HCN channels

I canali attivati dalla iperpolarizzazione e modulati da nucleotidi ciclici(HCN) sono una particolare classe di canali ionici voltaggio-dipendenti, che mediano la corrente If nel tessuto cardiaco e la Ih nel sistema nervoso centrale (SNC). Sono stati descritti per la prima volta nelle cellule pacemaker cardiache, dove rivestono un ruolo chiave sia nella generazione che nella regolazione del ritmo cardiaco. Nel SNC essi partecipano in processi quali l’integrazione dendritica, la sincronizzazione delle reti neurali, il potenziamento a lungo termine e la memorizzazione di compiti motori. A causa del loro ruolo cardiaco, i canali HCN rappresentano un target per la farmacologia clinica, ma purtroppo una percentuale dei pazienti trattati con inibitori degli HCN riportano effetti collaterali a livello visivo. Essi sono probabilmente dovuti all’interazione coi canali HCN presenti nella retina, dove sono espresse tutte e quattro le isoforme. Questa tesi indaga il ruolo dei canali HCN nella funzione visiva. La distribuzione dei canali HCN è stata valutata nella retina murina mediante tecniche immunoistochimiche. Il ruolo funzionale degli stessi nella trasmissione del segnale visivo è stato indagato in dettaglio mediante patch-clamp perforato su cellule bipolari dei bastoncelli (RBC) in sezioni retiniche, e tramite la tecnica non invasiva dell’elettroretinogramma (ERG) in ratti, topi wild type e topi knockout per il gene hcn1 (hcn1-ko). Le RBC esibiscono la Ih in risposta a step iperpolarizzanti. Laddove questa corrente è attiva, il profilo d’impedenza di queste cellule assume caratteristiche passa-banda. L’applizazione del bloccante HCN selettivo ZD7288 inibisce la Ih , trasformando il profilo d’impedenza in passa-basso. Questi comportamenti vengono predetti da un modello matematico della RBC dove si assume che Ih sia l’unica conduttanza di tipo attivo. Il modello predice anche che l’Ih è capace di accelerare la risposta alla luce nelle RBC. L’inibizione dei canali HCN in-vivo mediante somministrazione del bloccante Ivabradine induce un leggero prolungamento della risposta a flash di bassa intensità luminosa. Allo stesso tempo provoca una consistente modifica della relazione frequenza - ristposta da un profilo passa-banda ad uno passa-basso, sia a seguito di somministrazione acuta che prolungata; questi effetti sono reversibili mediante interruzione del trattamento. Nei topi hcn1-ko le risposte ERG da flash si presentano prolungate rispetto agli animali di controllo. In risposta a stimoli sinusoidali la delezione di hcn1 provoca una parziale attenuazione del comportamento passa banda, senza arrivare però ad un comportamento passa-basso come osservato dopo somministrazione del bloccante organico. Considerati nel loro insieme, questi dati mettono in luce l’importanza dei canali HCN nei primi stadi della trasmissione del segnale visivo dei bastoncelli. Fornendo così un bagaglio di conoscenze che presto potrebbe spiegare il ruolo della Ih negli effetti collaterali riportati durante l’uso clinico degli inibitori dei canali HCN. Gli esprimenti sugli hcn1-ko, inoltre, rappresentano la prima prova che il preservare anche una parziale funzionalità della Ih nella retina, possa essere una buona strategia da seguire per mitigare gli effetti collaterali che questi farmaci provocano a livello visivo

Involvement of Autophagic Pathway in the Progression of Retinal Degeneration in a Mouse Model of Diabetes

The notion that diabetic retinopathy (DR) is essentially a micro-vascular disease has been recently challenged by studies reporting that vascular changes are preceded by signs of damage and loss of retinal neurons. As to the mode by which neuronal death occurs, the evidence that apoptosis is the main cause of neuronal loss is far from compelling. The objective of this study was to investigate these controversies in a mouse model of streptozotocin (STZ) induced diabetes. Starting from 8 weeks after diabetes induction there was loss of rod but not of cone photoreceptors, together with reduced thickness of the outer and inner synaptic layers. Correspondingly, rhodopsin expression was downregulated and the scotopic electroretinogram (ERG) is suppressed. In contrast, cone opsin expression and photopic ERG response were not affected. Suppression of the scotopic ERG preceded morphological changes as well as any detectable sign of vascular alteration. Only sparse apoptotic figures were detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and glia was not activated. The physiological autophagy flow was altered instead, as seen by increased LC3 immunostaining at the level of outer plexiform layer (OPL) and upregulation of the autophagic proteins Beclin-1 and Atg5. Collectively, our results show that the streptozotocin induced DR in mouse initiates with a functional loss of the rod visual pathway. The pathogenic pathways leading to cell death develop with the initial dysregulation of autophagy well before the appearance of signs of vascular damage and without strong involvement of apoptosis

P-satI-D Shape Regulation of Soft Robots

Soft robots are intrinsically underactuated mechanical systems that operate under uncertainties and disturbances. In these conditions, this letter proposes two versions of PID-like control laws with a saturated integral action for the particularly challenging shape regulation task. The closed-loop system is asymptotically stabilized and matched constant disturbances are rejected using a very reduced amount of system information for control implementation. Stability is assessed on the underactuated dynamic model through the Invariant Set Theorem for two relevant classes of soft robots, i.e., elastically decoupled and elastically dominated soft robots. Extensive simulation results validate the proposed controllers

TMEM16A is associated with voltage-gated calcium channels in mouse retina and its function is disrupted upon mutation of the auxiliary α2δ4 subunit

Photoreceptors rely upon highly specialized synapses to efficiently transmit signals to multiple postsynaptic targets. Calcium influx in the presynaptic terminal is mediated by voltage-gated calcium channels (VGCC). This event triggers neurotransmitter release, but also gates calcium-activated chloride channels (TMEM), which in turn regulate VGCC activity. In order to investigate the relationship between VGCC and TMEM channels, we analyzed the retina of wild type (WT) and Cacna2d4 mutant mice, in which the VGCC auxiliary a2d4 subunit carries a nonsense mutation, disrupting the normal channel function. Synaptic terminals of mutant photoreceptors are disarranged and synaptic proteins as well as TMEM16A channels lose their characteristic localization. In parallel, calcium-activated chloride currents are impaired in rods, despite unaltered TMEM16A protein levels. Co-immunoprecipitation revealed the interaction between VGCC and TMEM16A channels in the retina. Heterologous expression of these channels in tsA-201 cells showed that TMEM16A associates with the CaV1.4 subunit, and the association persists upon expression of the mutant a2d4 subunit. Collectively, our experiments show association between TMEM16A and the a1 subunit of VGCC. Close proximity of these channels allows optimal function of the photoreceptor synaptic terminal under physiological conditions, but also makes TMEM16A channels susceptible to changes occurring to calcium channels

Processing of Retinal Signals in Normal and HCN Deficient Mice

This study investigates the role of two different HCN channel isoforms in the light response of the outer retina. Taking advantage of HCN-deficient mice models and of in vitro (patch-clamp) and in vivo (ERG) recordings of retinal activity we show that HCN1 and HCN2 channels are expressed at distinct retinal sites and serve different functions. Specifically, HCN1 operate mainly at the level of the photoreceptor inner segment from where, together with other voltage sensitive channels, they control the time course of the response to bright light. Conversely, HCN2 channels are mainly expressed on the dendrites of bipolar cells and affect the response to dim lights. Single cell recordings in HCN1−/− mice or during a pharmacological blockade of Ih show that, contrary to previous reports, Ikx alone is able to generate the fast initial transient in the rod bright flash response. Here we demonstrate that the relative contribution of Ih and Ikx to the rods' temporal tuning depends on the membrane potential. This is the first instance in which the light response of normal and HCN1- or HCN2-deficient mice is analyzed in single cells in retinal slice preparations and in integrated full field ERG responses from intact animals. This comparison reveals a high degree of correlation between single cell current clamp data and ERG measurements. A novel picture emerges showing that the temporal profile of the visual response to dim and bright luminance changes is separately determined by the coordinated gating of distinct voltage dependent conductances in photoreceptors and bipolar 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

Biolistic Labeling of Retinal Ganglion Cells

Labeling of cellular structures is of fundamental importance in the investigation of diseases of the central nervous system. Biolistic labeling of retinal ganglion cells (RGCs) allows visualization of dendritic and synaptic structures of RGCs in retinal explants from animal models of experimental glaucoma. This technique sparsely labels RGCs, and, due to the stochastic nature of the particle delivery, all RGC types can be potentially observed in the labeled tissue. Quantification of dendritic and synaptic properties permits examination of the specific alterations to RGC morphology at different stages of degeneration, such as dendritic shrinkage and excitatory synapse loss

Who's lost first? Susceptibility of retinal ganglion cell types in experimental glaucoma

The purpose of this article is to summarize our current knowledge about the susceptibility of specific retinal ganglion cell (RGC) types in experimental glaucoma, and to delineate the initial morphological and functional alterations that occur in response to intraocular pressure (IOP) elevation. There has been debate in the field as to whether RGCs with large somata and axons are more vulnerable, with definitive conclusions still in progress because of the wide diversity of RGC types. Indeed, it is now estimated that there are greater than 30 different RGC types, and while we do not yet understand the complete details, we discuss a growing body of work that supports the selective vulnerability hypothesis of specific RGC types in experimental glaucoma. Specifically, structural and functional degeneration of various RGC types have been examined across different rodent models of experimental glaucoma (acute vs. chronic) and different strains, and an emerging consensus is that OFF RGCs appear to be more vulnerable to IOP elevation compared to ON RGCs. Understanding the mechanisms by which this selective vulnerability manifests across different RGC types should lead to novel and improved strategies for neuroprotection and neuroregeneration in glaucoma