Capacitance measurement of dendritic exocytosis in an electrically coupled inhibitory retinal interneuron: an experimental and computational study

Exocytotic release of neurotransmitter can be quantified by electrophysiological recording from postsynaptic neurons. Alternatively, fusion of synaptic vesicles with the cell membrane can be measured as increased capacitance by recording directly from a presynaptic neuron. The “Sine + DC” technique is based on recording from an unbranched cell, represented by an electrically equivalent RC-circuit. It is challenging to extend such measurements to branching neurons where exocytosis occurs at a distance from a somatic recording electrode. The AII amacrine is an important inhibitory interneuron of the mammalian retina and there is evidence that exocytosis at presynaptic lobular dendrites increases the capacitance. Here, we combined electrophysiological recording and computer simulations with realistic compartmental models to explore capacitance measurements of rat AII amacrine cells. First, we verified the ability of the “Sine + DC” technique to detect depolarizationevoked exocytosis in physiological recordings. Next, we used compartmental modeling to demonstrate that capacitance measurements can detect increased membrane surface area at lobular dendrites. However, the accuracy declines for lobular dendrites located further from the soma due to frequency-dependent signal attenuation. For sine wave frequencies ≥1 kHz, the magnitude of the total releasable pool of synaptic vesicles will be significantly underestimated. Reducing the sine wave frequency increases overall accuracy, but when the frequency is sufficiently low that exocytosis can be detected with high accuracy from all lobular dendrites (~100 Hz), strong electrical coupling between AII amacrines compromises the measurements. These results need to be taken into account in studies with capacitance measurements from these and other electrically coupled neurons.publishedVersio

Studies on horizontal cells of the carp retina with special reference to temperature and calcium

Carp [Cyprinus carpio) were acclimated to 8±1 C, 16±1.5 C and 26±1 C. Dark adapted retinas were isolated and light induced responses of HI horizontal cells recorded. The dynamic range of these cells was affected by temperature, showing a decrease on heating or cooling from an optimum temperature. The effect of acclimation was to shift this optimum in an adaptive manner. A move from 16 C to 8 C resulted in ~44% acclimation, while a move from 16 C to 26 C resulted in ~67% acclimation. The rates of change of membrane potential and latency of the response also showed adaptive changes on acclimation. Isolated horizontal cells were voltage clamped using the whole cell patch clamp technique. The current-voltage (I-V) relationship of the prominent anomalous rectifier current was displaced by changes in the extracellular potassium concentration and was blocked by Ba(^2+) or Rb(^+). Its amplitude did not appear to be affected by thermal acclimation. A pharmacologically isolated sustained Ca(^2+) current, with an I-V relationship characteristic of an L-type current, also showed no apparent thermal acclimation. The ratiometric calcium indicator Fura-2 was used to measure the intracellular calcium concentration in isolated horizontal cells. The intracellular calcium concentration rose on depolarization of the cells, in an extracellular calcium concentration dependent manner. This increase was blocked by various metal ions with varying sensitivities: La(^3+)>Cd(^2+)>Cu(^2+)>Co≥Ni(^2+). The rate of change of intracellular calcium concentration was increased by increased temperature, but did not appear to be affected by thermal acclimation. Sustained depolarizations (up to 15 minutes) resulted in sustained elevations in intracellular calcium concentration proportional to the degree of depolarization. Possible mechanisms underlying the long and short term effects of temperature on the horizontal cell responses are discussed. The sustained calcium current and the intracellular calcium concentration changes are disscused in terms of the potential roles of this current and the significance of the subsequent intracellular calcium concentration changes

Electrical Communication and its Physiological Relevance in Retinal Pigment Epithelium

Verkkokalvon pigmenttiepiteeli (eng. retinal pigment epithelium, RPE) tekee tiivistä yhteistyötä verkkokalvon kanssa turvatakseen näköaistin toiminnan. Useita RPE:n tärkeimpiä tehtäviä, kuten valoa aistivien näköaistinsolujen uusiutumista, säädellään ionikanavien avulla. Näiden kanavien toimintaa ja RPE:n kykyä säädellä kalvopotentiaaliaan ei kuitenkaan vielä täysin ymmärretä. Tässä väitöskirjatyössä tutkittiin jänniteherkkien ionikanavien toimintaa sekä RPE:n sähköistä kytkeytyvyyttä käyttäen mallina ihmisen alkion kantasoluista erilaistettuja RPE-soluja sekä hiiren RPE-kudosta. Jänniteherkät natriumkanavat (NaV) tunnetaan parhaiten osallisuudestaan aktiopotentiaalin synnyssä hermosoluissa, mutta näiden kanavien tiedetään esiintyvän myös useissa muissa solutyypeissä, kuten makrofageissa ja astrosyyteissä, joissa aktiopotentiaaleja ei lähtökohtaisesti muodostu. NaV kanavien ei kuitenkaan uskottu esiintyvän RPE-kudoksessa huolimatta siitä, että niitä on toisinaan havaittu RPE-soluviljelmissä. Tämä väitöskirjatyö osoitti, että sekä kantasoluista erilaistetuissa RPE-soluissa että hiiren RPE-soluissa ilmentyy useita NaV- kanavaperheen alatyyppejä. Samalla havaittiin, että aikaisempi virheellinen johtopäätös aiheutui tutkimusten suorittamisesta yksittäisillä soluilla toiminnallisen kudoksen sijaan. Tässä työssä osoitettiin myös löydettyjen Nav-kanavatyyppien toiminnallisuus sähköfysiologisilla mittauksilla. Merkittävimpien kanavatyyppien (NaV1.4–NaV1.6 sekä NaV1.8) havaittiin sijoittuvan solu-soluliitoksiin tai RPE:n apikaaliselle solukalvolle. RPE-solujen sähköfysiogisia mittauksia on tyypillisesti tehty yksittäisistä eristetyistä soluista. Tästä johtuen RPE-solujen sähköistä kytkeytyvyyttä ei ole selvitetty nisäkkäillä, vaikka tiedetään, että aukkoliitoksilla on tärkeitä tehtäviä silmän kehityksessä. Tämä väitöskirjatyö osoitti, että solujen merkittävin aukkoliitosproteiini (engl. Connexin, Cx) on Cx43, jonka havaittiin muodostavan sekä aukkoliitoksia että puolikkaita hemikanavia solujen apikaalisella pinnalla. Sähköfysiologiset mittaukset osoittivat, että RPE:n laajasta aukkoliitosten verkostosta huolimatta RPE-solujen välinen kytkeytyvyys on suhteellisen alhainen. Kytkeytyvyyttä voitiin kuitenkin säädellä aukkoliitosten farmakologisilla estäjillä, tai estämällä tietyn Cdk5 (engl. cyclin-dependent kinase 5) kinaasi-entsyymin toimintaa. NaV-kanavien ja aukkoliitosten merkitystä RPE:n fysiologiassa tutkittiin keskittymällä näköaistinsolujen kalvojen uusiutumiseen, jossa RPE:n solusyönti eli fagosytoosi on merkittävässä roolissa. Tulokset osoittivat, että fagosytoosin aikana NaV-alatyypit NaV1.4 ja NaV1.8 esiintyvät lähellä näköaistinsolujen kalvopartikkeleita. NaV-kanavien toiminnan estäminen farmakologisesti tai geneettisesti (engl. short hairpin RNA, shRNA) vähensi merkittävästi fagosytoosin tehokkuutta. Lisäksi näiden kanavien havaittiin paikantuvan sekä apikaalipinnalle muodostuviin fagosytoosi-kuppeihin, että jo sisään otettuihin fagosomeihin yhdessä endosomien markkeriproteiinien (engl. rat sarcoma virus-related protein, Rab7) kanssa. Nämä tulokset antavat viitteitä siitä, että NaV-kanavilla olisi monipuolisia tehtäviä fagosytoosin aikana. NaV-kanavien lisäksi myös Cx43:n havaittiin esiintyvän näköaistinsolujen kalvopartikkelien kanssa fagosytoosissa ja tulokset antavat viitteitä siitä, että aukkoliitoksia otetaan solujen sisälle prosessin aikana. Fosforylaation havaittiin säätelevän tätä aukkoliitosten siirtymää ja erityisesti Cdk5-, ja proteiinikinaasi C- entsyymeillä oli merkittävä rooli tässä säätelyssä. Tämän työn tulokset osoittivat, että Cx43 liittyy fagosytoosikuppien muodostukseen sekä solujen aktiini-tukirangan uudelleen järjestymiseen. Fagosytoosin säätelyn tiedetään perustuvan vuorokausirytmiin ja mielenkiintoista on, että Cdk5-kinaasin on osoitettu vaikuttavan tähän rytmiin. On siis mahdollista, että Cdk5 auttaa myös fagosytoosin ajoituksen säätelyssä. Kokonaisuutena työni osoittaa RPE:n fysiologian ja sen ionikanavakoneiston säätelyn monimutkaisuuden. Nav-kanavien roolin on havaittu olevan huomattavasti monipuolisempi kuin aktiopotentiaalien muodostus hermosoluissa ja tuloksemme vahvistavat tätä käsitystä. Yksi työni yllättävimmistä ja merkittävimmistä tuloksista oli, että RPE voi säädellä kalvojännitettään ja epiteelikudoksen solujen välistä viestintäänsä nopeasti. Tarkemmat tiedot tämän ionisignaloinnin roolista fagosytoosissa lisäävät ymmärrystämme prosessista, joka on merkittävä näkökyvyllemme. Yhteenvetona tämä työ osoittaa, että RPE:n rooli yhteistyössä verkkokalvon kanssa on paljon aktiivisempi kuin on aikaisemmin luultu.Retinal pigment epithelium (RPE) is a tissue that preserves the health and functionality of its closely associated neural tissue, the retina. Many of the essential functions of RPE, including the renewal of light-sensing retinal photoreceptors, are regulated by ion channels. Yet, the involved ionic mechanisms, the extent of membrane potential dynamics, and the intercellular communication are not entirely understood. In this thesis, I studied the voltage-gated ion channels and electrical coupling of RPE in both human embryonic stem cell-derived and mouse RPE. Voltage-gated sodium channels (NaV), while best known for their role in action potential generation, are expressed in several non-excitable cell types such as macrophages and astrocytes. Yet, these channels had not been considered to exist in native RPE, although they had occasionally been detected in cell culture. This thesis demonstrates that stem cell-derived and mouse RPE exhibit several subtypes of NaV channels and that their earlier dismissal was due to cell isolation procedures. Our electrophysiological recordings showed that these identified NaV channels are functional. The main channel subtypes NaV1.4–NaV1.6 and NaV1.8 were found to localize in the cell-cell junctions or apical membrane in RPE. As the conventional method to carry out electrophysiological recordings in RPE is to use single cells, the electrical connectivity had not been characterized in mammalian RPE, despite the importance of gap junctions in ocular development. In this thesis, we showed that the major connexin (Cx) isoform was Cx43 which was found to form both gap junctions and apical hemichannels. The electrophysiological recordings demonstrated that the electrical connectivity was relatively low despite the extensive network of gap junctions in RPE. Yet, it was modifiable by gap junction blockers or by inhibiting a specific kinase known as cyclin-dependent kinase 5 (Cdk5). The significance of NaV channels and gap junctions to RPE physiology was investigated by focusing on the renewal of photoreceptor outer segments, where phagocytosis by RPE plays a key role. The results demonstrated that NaV subtypes NaV1.4 and NaV1.8 localize with the outer segments during phagocytosis. Moreover, inhibiting the activity of NaV channels with pharmacological modulators or short hairpin RNA (shRNA) significantly impaired phagocytosis efficiency. Furthermore, Nav channels were found to localize to the forming phagocytic cups in the apical membrane and the ingested phagosomes together with an endosomal marker Rab7. The results obtained in this thesis imply that NaV channels have versatile roles in phagocytosis. In addition to NaV channels, Cx43 localized adjacent to outer segments during phagocytosis, and the results indicate that gap junctions are internalized during the process. This translocation of gap junctions was shown to be regulated by phosphorylation, particularly by kinases such as Cdk5 and protein kinase C. The results obtained in this thesis imply that Cx43 is involved in the formation of phagocytic cups. As phagocytosis is known to be under circadian control, and Cdk5 has previously been shown to regulate this cycle, it is plausible that Cdk5 helps to control the rhythm of photoreceptor renewal. Our results highlight the complexity of RPE physiology and its ion channel machinery. The findings add to the growing body of evidence demonstrating that NaV channels' role is much more diverse than action potential generation. The results show that RPE can generate fast changes in voltage and rapidly modify its cell-cell connectivity across the epithelium. Gaining a deeper understanding of the involvement of ionic mechanisms in phagocytosis could help us to understand the phagocytosis pathway both in the healthy and diseased eye. Ultimately, this work highlights that RPE's role in its interaction with the neural retina is far more active than was previously thought

Astrocytic Gq-GPCR-Linked IP3R-Dependent Ca2+ Signaling Does Not Mediate Neurovascular Coupling in Mouse Visual Cortex in vivo

Local blood flow is modulated in response to changing patterns of neuronal activity (Roy and Sherrington, 1890), a process termed neurovascular coupling. It has been proposed that the central cellular pathway driving this process is astrocytic Gq-GPCR-linked IP3R-dependent Ca2+ signaling, though in vivo tests of this hypothesis are largely lacking. We examined the impact of astrocytic Gq-GPCR and IP3R-dependent Ca2+ signaling on cortical blood flow in awake, responsive mice using multiphoton laser-scanning microscopy and novel genetic tools that enable the selective manipulation of astrocytic signaling pathways in vivo. Selective stimulation of astrocytic Gq-GPCR cascades and downstream Ca2+ signaling with the hM3Dq DREADD designer receptor system was insufficient to modulate basal cortical blood flow. We found no evidence of observable astrocyte endfeet Ca2+ elevations following physiological visual stimulation despite robust dilations of adjacent arterioles using cyto-GCaMP3 and Lck-GCaMP6s, the most sensitive Ca2+ indicator available. Astrocytic Ca2+ elevations could be evoked when inducing the startle response with unexpected air puffs. However, startle-induced astrocytic Ca2+ signals did not precede corresponding startle-induced hemodynamic changes. Further, neurovascular coupling was intact in awake, responsive mice genetically lacking astrocytic IP3R-dependent Ca2+ signaling (IP3R2 KO). These data establish that astrocytic Gq-GPCR-linked IP3R-dependent Ca2+ signaling does not mediate neurovascular coupling in visual cortex of awake, responsive mice.Doctor of Philosoph

Exploring the Cellular and Molecular Mechanisms Driving Connexin-36 Electrical Plasticity and the Functional Consequences of its Loss on the Sensorimotor Behaviors of Zebrafish (Danio Rerio)

In mammals, connexin-36 (Cx36) is the major component of electrical synapses, also found alongside chemical synapses throughout the brain. An equivalent form of long-term potentiation (LTP) exclusively at Cx36 electrical synapses, termed the run-up, has been previously identified; however, the mechanism and molecular machinery involved remains elusive. We hypothesized an LTP-like mechanism was involved at Cx36 electrical synapses, potentiating the run-up. To address this, we investigated the tubulin-dependent delivery of Cx36 connexons to the plasma membrane in Neuro2a cells. A putative Cx36-tubulin binding motif was elucidated via sequence alignment, and the direct binding of tubulin with the C-terminal tail of Cx36 was confirmed via BioID. TIRF and FRAP microscopy techniques established that Cx36 is transported from the trans-Golgi network to the plasma membrane via interactions with the tubulin-cytoskeleton. Manipulating the Cx36-tubulin interaction via mutations and pharmacological inhibition reduced Cx36 trafficking to the gap junction plaque and inhibited run-up. While we identified tubulin as a driving force in achieving Cx36 run-up, we found that its transport occurred independently of CaM-CaMKII activity. Despite the similarities in machinery used between chemical and electrical synapses, the functional consequence of Cx36 asymmetry at homotypic aggregates had yet to be addressed. Next, we investigated the role of Cx36 in sensorimotor circuitry in zebrafish (Danio rerio), where the teleost orthologs are expressed distinctly. Here, we targeted the gjd2b gene since its corresponding ortholog, Cx35b, is expressed presynaptically. Based on current literature, we hypothesized that complex alterations to neuronal circuitry via Cx35b knock-out (KO) would affect vision, sensorimotor gating, and plasticity-dependent cognitive processing. We found that Cx35b expression coincides with photoreceptor cell specificity and expression in the inner plexiform layer. Cx35b KO resulted in developmental deviances, cranial defects, visual ambiguities (discrimination of light stimulus brightness, arrangement, and temporal properties), and increased levels of anxiety; however, KO did not impair non-associative learning and memory. Genetically, loss of the gjd2b gene resulted in changes primarily in connexin, dopaminergic, and immediate early gene regulation. Taken together, we concluded that Cx36 shares molecular machinery with chemical synapses to rectify neuronal communication, and this molecular asymmetry is critical in driving behavioral outcomes

Exploring the Cellular and Molecular Mechanisms Driving Connexin-36 Electrical Plasticity and the Functional Consequences of its Loss on the Sensorimotor Behaviors of Zebrafish (Danio Rerio)

In mammals, connexin-36 (Cx36) is the major component of electrical synapses, also found alongside chemical synapses throughout the brain. An equivalent form of long-term potentiation (LTP) exclusively at Cx36 electrical synapses, termed the run-up, has been previously identified; however, the mechanism and molecular machinery involved remains elusive. We hypothesized an LTP-like mechanism was involved at Cx36 electrical synapses, potentiating the run-up. To address this, we investigated the tubulin-dependent delivery of Cx36 connexons to the plasma membrane in Neuro2a cells. A putative Cx36-tubulin binding motif was elucidated via sequence alignment, and the direct binding of tubulin with the C-terminal tail of Cx36 was confirmed via BioID. TIRF and FRAP microscopy techniques established that Cx36 is transported from the trans-Golgi network to the plasma membrane via interactions with the tubulin-cytoskeleton. Manipulating the Cx36-tubulin interaction via mutations and pharmacological inhibition reduced Cx36 trafficking to the gap junction plaque and inhibited run-up. While we identified tubulin as a driving force in achieving Cx36 run-up, we found that its transport occurred independently of CaM-CaMKII activity. Despite the similarities in machinery used between chemical and electrical synapses, the functional consequence of Cx36 asymmetry at homotypic aggregates had yet to be addressed. Next, we investigated the role of Cx36 in sensorimotor circuitry in zebrafish (Danio rerio), where the teleost orthologs are expressed distinctly. Here, we targeted the gjd2b gene since its corresponding ortholog, Cx35b, is expressed presynaptically. Based on current literature, we hypothesized that complex alterations to neuronal circuitry via Cx35b knock-out (KO) would affect vision, sensorimotor gating, and plasticity-dependent cognitive processing. We found that Cx35b expression coincides with photoreceptor cell specificity and expression in the inner plexiform layer. Cx35b KO resulted in developmental deviances, cranial defects, visual ambiguities (discrimination of light stimulus brightness, arrangement, and temporal properties), and increased levels of anxiety; however, KO did not impair non-associative learning and memory. Genetically, loss of the gjd2b gene resulted in changes primarily in connexin, dopaminergic, and immediate early gene regulation. Taken together, we concluded that Cx36 shares molecular machinery with chemical synapses to rectify neuronal communication, and this molecular asymmetry is critical in driving behavioral outcomes

A Novel Mechanism for Switching a Neural System from One State to Another

An animal's ability to rapidly adjust to new conditions is essential to its survival. The nervous system, then, must be built with the flexibility to adjust, or shift, its processing capabilities on the fly. To understand how this flexibility comes about, we tracked a well-known behavioral shift, a visual integration shift, down to its underlying circuitry, and found that it is produced by a novel mechanism – a change in gap junction coupling that can turn a cell class on and off. The results showed that the turning on and off of a cell class shifted the circuit's behavior from one state to another, and, likewise, the animal's behavior. The widespread presence of similar gap junction-coupled networks in the brain suggests that this mechanism may underlie other behavioral shifts as well

Vertebrate vision : about physical determinants of photoreceptor sensitivity and kinetics

Rod and cone photoreceptors transform information about incoming light into neural signals with broadly similar molecular mechanisms. Yet their sensitivity, response kinetics and adaptation properties are quite different as rods mediate dim-light vision and cones function mainly under daylight. This thesis 1. addresses the functional differences between rods and cones as well as mammalian and non-mammalian photoreceptors and 2. provides novel findings regarding the existence and regulation of rod-cone interactions at the photoreceptor level. Rod and cone photoresponses to brief flashes of light were recorded with electroretinogram (ERG) from isolated rodent and amphibian retinas. Various phototransduction models were used to compare their relevant parameters over a range of adapting conditions. The study focused on how the following physical factors shape and limit photoreceptor function: operating temperature, thermal stability of the amphibian long wavelength sensitive (A1-)visual pigment, outer segment dimensions, morphology and electrical connections between adjacent rods and cones. Mammalian rod photoreceptors generate faster photoresponses but light-adapt less efficiently than amphibian rods. In the rodent and anuran rods studied in this thesis, the main differences could be accounted for by the higher operating temperature and smaller outer segment size of the rodent photoreceptors. Additionally, the slender outer segments of the mammalian rods enabled sufficient quantal responses and high quantum catch despite the observed desensitizing effect of warming. Long wavelength -sensitive cone photoreceptors have been hypothesized to be desensitized by thermal excitation of their visual pigment molecules. However, it has been shown experimentally only in amphibian cones that utilize the A2-chromophore. The relative stability of the A1-based cone pigments - used by all terrestrial vertebrates - has remained unclear, as well as its role in limiting cone function. In this study, thermal isomerization rate of the long wavelength sensitive (A1-)visual pigment was estimated to play at most a minor role in regulating cone sensitivity of the frog Rana temporaria. Finally, ERG light responses originating in mouse cone photoreceptors were found to be suppressed in the dark-adapted retina, apparently through direct electrical coupling between rods and cones. The results indicated this coupling is weakened by moderate background light, explaining a long known phenomenon of unknown origin: light-induced growth of cone flash responses in mammalian ERG. This is indicative of a previously unknown mechanism of retinal adaptation