Computational modeling of spike generation in serotonergic neurons of the dorsal raphe nucleu

We consider here a single-compartment model of these neurons which is capable of describing many of the known features of spike generation, particularly the slow rhythmic pacemaking activity often observed in these cells in a variety of species. Included in the model are ten kinds of voltage dependent ion channels as well as calcium-dependent potassium current. Calcium dynamics includes buffering and pumping. In sections 3-9, each component is considered in detail and parameters estimated from voltage clamp data where possible. In the next two sections simplified versions of some components are employed to explore the effects of various parameters on spiking, using a systematic approach, ending up with the following eleven components: a fast sodium current $I_{Na}$, a delayed rectifier potassium current $I_{KDR}$, a transient potassium current $I_A$, a low-threshold calcium current $I_T$, two high threshold calcium currents $I_L$ and $I_N$, small and large conductance potassium currents $I_{SK}$ and $I_{BK}$, a hyperpolarization-activated cation current $I_H$, a leak current $I_{Leak}$ and intracellular calcium ion concentration $Ca_i$. Attention is focused on the properties usually associated with these neurons, particularly long duration of action potential, pacemaker-like spiking and the ramp-like return to threshold after a spike. In some cases the membrane potential trajectories display doublets or have kinks or notches as have been reported in some experimental studies. The computed time courses of $I_A$ and $I_T$ during the interspike interval support the generally held view of a competition between them in influencing the frequency of spiking. Spontaneous spiking could be obtained with small changes in a few parameters from their values with driven spiking.Comment: The abstract has been truncate

Kv2.1 mediates spatial and functional coupling of L-type calcium channels and ryanodine receptors in mammalian neurons.

The voltage-gated K+ channel Kv2.1 serves a major structural role in the soma and proximal dendrites of mammalian brain neurons, tethering the plasma membrane (PM) to endoplasmic reticulum (ER). Although Kv2.1 clustering at neuronal ER-PM junctions (EPJs) is tightly regulated and highly conserved, its function remains unclear. By identifying and evaluating proteins in close spatial proximity to Kv2.1-containing EPJs, we discovered that a significant role of Kv2.1 at EPJs is to promote the clustering and functional coupling of PM L-type Ca2+ channels (LTCCs) to ryanodine receptor (RyR) ER Ca2+ release channels. Kv2.1 clustering also unexpectedly enhanced LTCC opening at polarized membrane potentials. This enabled Kv2.1-LTCC-RyR triads to generate localized Ca2+ release events (i.e., Ca2+ sparks) independently of action potentials. Together, these findings uncover a novel mode of LTCC regulation and establish a unique mechanism whereby Kv2.1-associated EPJs provide a molecular platform for localized somatodendritic Ca2+ signals in mammalian brain neurons

Multiscale Model of Cerebral Blood Flow Control: Application to Small Vessel Disease and Cortical Spreading Depression

An in-time delivery of oxygen-rich blood into areas of high metabolic demand is pivotal in proper functioning of the brain and neuronal health. This highly precise communication between neuronal activity and cerebral blood flow (CBF) is termed as neurovascular coupling (NVC) or functional hyperemia. NVC is disrupted in major pathological conditions including Alzheimer’s disease, dementia, small vessel pathologies (SVD) and cortical spreading depression. Despite the utmost importance of NVC, its underlying mechanisms are not fully understood. This dissertation presents a multiscale mathematical modeling framework for studying unresolved mechanisms of NVC with major focus on K+ ions as a mediator of this process. To this end, models of single-cell electrophysiology are developed for endothelial (EC) and smooth muscle (SMC) cells of capillaries and parenchymal arterioles (PAs). Cells are electrically coupled, and large-scale geometrically-accurate models of microvascular networks are constructed. Model simulations predict an important role of capillary inward rectifying potassium channels (Kir) to sense neuronally-induced changes in extracellular potassium concentrations ([K+]o) and conduct hyperpolarizing signals over long distances to upstream PAs. Simulation results demonstrate a “tug-of-war” dynamic between Kir and voltage-gated potassium (Kv) channels in determining the Vm and myogenic tone of PA SMCs during NVC in SVD. Results also predict a key role of Kir channels in the experimentally observed multiphasic vascular response during high elevations of [K+]o in cortical spreading depression. The multiscale models presented in this study were able to accurately capture several experimentally observed responses during NVC and provided insights into their potential underlying mechanisms in health and disease. These models provide a theoretical platform where macroscale, tissue-level responses can be related to microscale, single-cell signaling pathways

Mechanisms of ANO1 channel activation in sensory neurons

ANO1 (TMEM16A) is a Ca2+ activated Cl- channel (CaCC) expressed in peripheral somatosensory neurons responding to painful (noxious) stimuli. Previously, our lab has been able to demonstrate specific coupling of ANO1 to inositol 1,4,5-trisphosphate receptor (IP3R)-mediated Ca2+ release from the endoplasmic reticulum (ER) via G-protein coupled receptor (GPCR) activation. This phenomenon underscores excitatory and noxious effects of some mediators of inflammatory pain, such as pro-algesic and vasoactive neuropeptide bradykinin. To further investigate mechanisms of ANO1 activation in somatosensory neurons, I developed a dual imaging approach, which involved transfecting dorsal root ganglion (DRG) neurons with a halide sensitive EYFP mutant (H148Q/I152L) and simultaneous Ca2+ imaging to monitor CaCC activity. This methodology was successfully used to demonstrate robust coupling of CaCC activity to IP3R activation produced by bradykinin. Blockade of ANO1 using a selective inhibitor (T16A-inhA01) abolished CaCC activity induced by bradykinin application. In contrast to the ER-induced Ca2+ release, Ca2+ influx produced by depolarisation-induced activation of voltage gated Ca2+ channels (VGCCs) was relatively ineffective in activating ANO1, which is in good agreement with previous studies. TRPV1 activation by capsaicin was able to induce robust CaCC activity. Given the ability of TRPV1 to activate PLC isoforms and produce IP3, I further tested the mechanism by which ANO1 is activated by TRPV1. Depletion of the ER Ca2+ stores severely reduced both, the capsaicin-induced Ca2+ signals and the concurrent CaCC activation. Intriguingly, under extracellular Ca2+ free conditions capsaicin was still able to induce [Ca2+]i elevation, further illustrating the ability of TRPV1 to induce intracellular Ca2+ release. Finally, monitoring of ER specific-Ca2+ dynamics concurrently with CaCC activity unambiguously confirmed the ability of TRPV1 to produce ER-Ca2+ mobilisation. Importantly, IP3R blockade with xestospongin C reduced CaCC activity after TRPV1 activation. Collectively, these experiments suggest that a significant fraction of Ca2+ required for activation of ANO1 downstream of TRPV1 is indeed delivered through IP3R activation. Using ‘in-situ proteomics’ and super-resolution microscopy I investigated multi-protein complexes in ER-plasma membrane (ER-PM) junctions of DRG neurons involving ANO1, TRPV1 and IP3R1. I found using proximity ligation assay that all 3 proteins were within 40nm of each other; however there was a greater number of ANO1 and TRPV1 complexes compared to TRPV1/ANO1 and TRPV1/IP3R1 complexes. Two-colour stochastic optical reconstruction microscopy (STORM) was able to confirm these findings and demonstrate that there is indeed a greater percentage of complexes involving ANO1 and TRPV1. Preliminary triple-colour STORM suggested the presence of ANO1, TRPV1 and IP3R1-containing protein complexes. Finally, I used total internal reflection microscopy (TIRF) to monitor the dynamics of the ER-PM junctions following the activation of bradykinin receptors or TRPV1. Application of bradykinin and capsaicin elicited increased intensity proximity of the ER to PM (as evaluated by the TIRF signal of fluorescently-labelled ER), which is suggestive of the ER moving to the PM by internal store mobilisation and highlighting the importance of ER-PM junctions. In sum, the experiments described in this thesis have discovered and characterised a novel mode of ANO1 activation in pain-sensing neurons: TRPV1-mediated ER Ca2+ release in ER-PM junctional signalling complex. These findings describe a hitherto unknown signalling mechanism potentially contributing to inflammatory pain

Arrhythmogenic late Ca2+sparks in failing heart cells and their control by action potential configuration

Sudden death in heart failure patients is a major clinical problem worldwide, but it is unclear how arrhythmogenic early afterdepolarizations (EADs) are triggered in failing heart cells. To examine EAD initiation, high-sensitivity intracellular Ca2+ measurements were combined with action potential voltage clamp techniques in a physiologically relevant heart failure model. In failing cells, the loss of Ca2+ release synchrony at the start of the action potential leads to an increase in number of microscopic intracellular Ca2+ release events (“late” Ca2+ sparks) during phase 2–3 of the action potential. These late Ca2+ sparks prolong the Ca2+ transient that activates contraction and can trigger propagating microscopic Ca2+ ripples, larger macroscopic Ca2+ waves, and EADs. Modification of the action potential to include steps to different potentials revealed the amount of current generated by these late Ca2+ sparks and their (subsequent) spatiotemporal summation into Ca2+ ripples/waves. Comparison of this current to the net current that causes action potential repolarization shows that late Ca2+ sparks provide a mechanism for EAD initiation. Computer simulations confirmed that this forms the basis of a strong oscillatory positive feedback system that can act in parallel with other purely voltage-dependent ionic mechanisms for EAD initiation. In failing heart cells, restoration of the action potential to a nonfailing phase 1 configuration improved the synchrony of excitation–contraction coupling, increased Ca2+ transient amplitude, and suppressed late Ca2+ sparks. Therapeutic control of late Ca2+ spark activity may provide an additional approach for treating heart failure and reduce the risk for sudden cardiac death

Engineering aequorin as an indicator of calcium signals near the BK channel

The BK channel is a large conductance calcium-activated voltage- dependent potassium channel. This channel plays a key role as a negative feedback mechanism of membrane excitability and cellular Ca2+. There is substantial evidence suggesting that the Ca2+ activation of the BK channel is regulated by localised Ca2+ release from intracellular stores. The aim of the work presented in this thesis was to develop a novel method of measuring the local Ca2+ concentration controlling the BK channel activation. The p2 subunit, an auxiliary protein of the BK channel, was extracted from MG63 cells and cloned. Subsequently, the aequorin sequence was attached to its C-terminus using splicing by overlapping extension. The recombinant protein retained the features of the native proteins emitting light in response to Ca2+ and showed correct targeting to the ceil membrane. The resultant light emission of the new protein was diminished in comparison to the native aequorin. The p2-Aequorin and a cytosolic Luciferase-aequorin were successfully transfected in a HEK293 cell line which stably express the BK channel a subunit. The expression of the aequorin constructs in HEK293 cells in suspension revealed the presence of intracellular mechanosensitive Ca2+ channels. The main finding of this thesis was that the Ca2+ affecting the BK channel is regulated independently of cytosolic Ca2+ in HEK293 cells. Stimulation with agonists such as carbachol, ATP and cyclopiazonic acid demonstrated clear differences in the magnitude of BK channel microdomain and cytosolic Ca2+ signals. Short term exposure to caffeine induced a significant decrease in the Ca2+ signals near the channel. The addition of extracellular Ca2+ led to large Ca2+ transients close to the BK channel suggesting a store-operated Ca2+ mechanism. The Ca2+ effects produced by carbachol, ATP, caffeine and cyclopizaonic acid indicate a coupling between IP3-induced Ca2+ release from the ER and Ca2+- activation of the BK channel.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Modulation of intrinsic and synaptic excitability during sleep oscillations and electrographic seizures

Le présente mémoire fournit des nouvelles évidences montrant la modulation de l’excitabilité neuronale intrinsèque et synaptique, et la conséquence de cette modulation sur l’activité neuronale durant à la fois, les oscillations lentes du sommeil, et les crises électrographiques in vivo chez des animaux anesthésiés. Nous effectuons des enregistrements intracellulaires simultanés de neurones corticaux et des potentiels de champs locaux au niveau du gyrus suprasylvien à l’intérieur du cortex associatif pariétal (aires : 5, 7 et 21). Nous suggérons que la fluctuation de la concentration extracellulaire du calcium durant les oscillations lentes du sommeil module à la fois, l’excitabilité intrinsèque et synaptique des neurones corticaux, ainsi par conséquent, elle module affecte la relation d’input-output de ces neurones. L’apparition durant les oscillations lentes du sommeil, des crises de type Lennex-Gastaut qui sont générées corticalement, nous a permet d’étudier les propriétés spatio-temporelles des ondes paroxysmiques rapides associées avec ce type de crises. Nous suggérons que les ondes paroxysmiques rapides apparaissent comme des oscillations quasi-indépendantes même dans les localisations corticales voisines, suggérant leur origine focal.The present memoir provides new evidences showing the modulation of intrinsic and synaptic excitability of cortical neurons, and the consequence of this modulation on neuronal activity during both slow sleep oscillations and electrographic seizures in vivo in anaesthetized animals. We performed simultaneous recordings of cortical neurons with local field potentials in suprasylvian gyrus within parietal associative cortex (area 5, 7 and 21). We suggest that the fluctuation of extacellular calcium concentration during slow sleep oscillations, modulates both intrinsic and synaptic excitability cortical neurons, thus by consequence modulates the input-output relationship of these neurons. The occurrence during slow-wave sleep of cortically generated Lennox-Gastaut type of seizures admits us to study the spatio-temporal properties of paroxysmal fast runs associated with this type of seizures. We suggest that fast runs appeared as quasi-independent oscillations even in neighbouring cortical locations suggesting their focal origin

Engineering aequorin as an indicator of calcium signals near the BK channel

The BK channel is a large conductance calcium-activated voltage- dependent potassium channel. This channel plays a key role as a negative feedback mechanism of membrane excitability and cellular Ca2+. There is substantial evidence suggesting that the Ca2+ activation of the BK channel is regulated by localised Ca2+ release from intracellular stores. The aim of the work presented in this thesis was to develop a novel method of measuring the local Ca2+ concentration controlling the BK channel activation. The p2 subunit, an auxiliary protein of the BK channel, was extracted from MG63 cells and cloned. Subsequently, the aequorin sequence was attached to its C-terminus using splicing by overlapping extension. The recombinant protein retained the features of the native proteins emitting light in response to Ca2+ and showed correct targeting to the ceil membrane. The resultant light emission of the new protein was diminished in comparison to the native aequorin. The p2-Aequorin and a cytosolic Luciferase-aequorin were successfully transfected in a HEK293 cell line which stably express the BK channel a subunit. The expression of the aequorin constructs in HEK293 cells in suspension revealed the presence of intracellular mechanosensitive Ca2+ channels. The main finding of this thesis was that the Ca2+ affecting the BK channel is regulated independently of cytosolic Ca2+ in HEK293 cells. Stimulation with agonists such as carbachol, ATP and cyclopiazonic acid demonstrated clear differences in the magnitude of BK channel microdomain and cytosolic Ca2+ signals. Short term exposure to caffeine induced a significant decrease in the Ca2+ signals near the channel. The addition of extracellular Ca2+ led to large Ca2+ transients close to the BK channel suggesting a store-operated Ca2+ mechanism. The Ca2+ effects produced by carbachol, ATP, caffeine and cyclopizaonic acid indicate a coupling between IP3-induced Ca2+ release from the ER and Ca2+- activation of the BK channel