The Influence of Ca2+ Buffers on Free [Ca2+] Fluctuations and the Effective Volume of Ca2+ Microdomains

Intracellular calcium (Ca2+) plays a significant role in many cell signaling pathways, some of which are localized to spatially restricted microdomains. Ca2+ binding proteins (Ca2+ buffers) play an important role in regulating Ca2+ concentration ([Ca2+]). Buffers typically slow [Ca2+] temporal dynamics and increase the effective volume of Ca2+ domains. Because fluctuations in [Oa(2+)] decrease in proportion to the square-root of a domain\u27s physical volume, one might conjecture that buffers decrease [Ca2+] fluctuations and, consequently, mitigate the significance of small domain volume concerning Ca2+ signaling. We test this hypothesis through mathematical and computational analysis of idealized buffer-containing domains and their stochastic dynamics during free Ca2+ influx with passive exchange of both Ca2+ and buffer with bulk concentrations. We derive Langevin equations for the fluctuating dynamics of Ca2+ and buffer and use these stochastic differential equations to determine the magnitude of [Ca2+] fluctuations for different buffer parameters (e.g., dissociation constant and concentration). In marked contrast to expectations based on a naive application of the principle of effective volume as employed in deterministic models of Ca2+ signaling, we find that mobile and rapid buffers typically increase the magnitude of domain [Ca2+] fluctuations during periods of Ca2+ influx, whereas stationary (immobile) Ca2+ buffers do not. Also contrary to expectations, we find that in the absence of Ca2+ influx, buffers influence the temporal characteristics, but not the magnitude, of [Ca2+] fluctuations. We derive an analytical formula describing the influence of rapid Ca2+ buffers on [Ca2+] fluctuations and, importantly, identify the stochastic analog of (deterministic) effective domain volume. Our results demonstrate that Ca2+ buffers alter the dynamics of [Ca2+] fluctuations in a nonintuitive manner. The finding that Ca2+ buffers do not suppress intrinsic domain [Ca2+] fluctuations raises the intriguing question of whether or not [Ca2+] fluctuations are a physiologically significant aspect of local Ca2+ signaling

Rôle de deux groupes de vésicules dans la transmission synaptique

Les synapses formées par les fibres moussues (FM) sur les cellules principales de la région CA3 (FM-CA3) jouent un rôle crucial pour la formation de la mémoire spatiale dans l’hippocampe. Une caractéristique des FM est la grande quantité de zinc localisée avec le glutamate dans les vésicules synaptiques recyclées par la voie d’endocytose dépendante de l’AP3. En combinant l’imagerie calcique et l’électrophysiologie, nous avons étudié le rôle des vésicules contenant le zinc dans la neurotransmission aux synapses FM-CA3. Contrairement aux études précédentes, nous n’avons pas observé de rôle pour le zinc dans l’induction des vagues calciques. Nos expériences ont révélé que les vagues calciques sont dépendantes de l’activation des récepteurs métabotropiques et ionotropiques du glutamate. D’autre part, nos données indiquent que les vésicules dérivées de la voie dépendante de l’AP3 forment un groupe de vésicules possédant des propriétés spécifiques. Elles contribuent principalement au relâchement asynchrone du glutamate. Ainsi, les cellules principales du CA3 de souris n’exprimant pas la protéine AP3 avaient une probabilité inférieure de décharge et une réduction de la synchronie des potentiels d’action lors de la stimulation à fréquences physiologiques. Cette diminution de la synchronie n’était pas associée avec un changement des paramètres quantiques ou de la taille des groupes de vésicules. Ces résultats supportent l’hypothèse que deux groupes de vésicules sont présents dans le même bouton synaptique. Le premier groupe est composé de vésicules recyclées par la voie d’endocytose utilisant la clathrine et participe au relâchement synchrone du glutamate. Le second groupe est constitué de vésicules ayant été recyclées par la voie d’endocytose dépendante de l’AP3 et contribue au relâchement asynchrone du glutamate. Ces deux groupes de vésicules sont nécessaires pour l’encodage de l’information et pourraient être importants pour la formation de la mémoire. Ainsi, les décharges de courte durée à haute fréquence observées lorsque les animaux pénètrent dans les places fields pourraient causer le relâchement asynchrone de glutamate. Finalement, les résultats de mon projet de doctorat valident l’existence et l’importance de deux groupes de vésicules dans les MF qui sont recyclées par des voies d’endocytoses distinctes et relâchées durant différents types d’activités.Mossy fiber-CA3 pyramidal cell synapses play a crucial role in the hippocampal formation of spatial memories. These synaptic connections possess a number of unique features substantial for its role in the information processing and coding. One of these features is presence of zinc co-localized with glutamate within a subpopulation of synaptic vesicles recycling through AP3-dependent bulk endocytosis. Using Ca2+ imaging and electrophysiological recordings we investigated role of these zinc containing vesicles in the neurotransmission. In contrast to previous reports, we did not observe any significant role of vesicular zinc in the induction of large postsynaptic Ca2+ waves triggered by burst stimulation. Moreover, our experiments revealed that Ca2+ waves mediated by Ca2+ release from internal stores are dependent not only on the activation of metabotropic, but also ionotropic glutamate receptors. Nevertheless, subsequent experiments unveiled that the vesicles derived via AP3-dependent endocytosis primary contribute to the asynchronous, but not synchronous mode of glutamate release. Futhermore, knockout mice lacking adaptor protein AP3 had a reduced synchronization of postsynaptic action potentials and impaired information transfer; this was not associated with any changes in the synchronous release quantal parameters and vesicle pool size. These findings strongly support the idea that within a single presynaptic bouton two heterogeneous pools of releasable vesicles are present. One pool of readily releasable vesicles forms via clathrin mediated endocytosis and mainly participates in the synchronous release; a second pool forms through bulk endocytosis and primarily supplies asynchronous release. The existence of two specialized pools is essential for the information coding and transfer within hippocampus. It also might be important for hippocampal memory formation. In contrast to low firing rates at rest, dentate gyrus granule cells tend to fire high frequency bursts once an animal enters a place field. These burst activities, embedded in the lower gamma frequency, should be especially efficient in the triggering of substantial asynchronous glutamate release. Therefore, the results of my PhD project for the first time provide strong evidence for the presence and physiological importance of two vesicle pools with heterogeneous release and recycling properties via separate endocytic pathways within the same mossy fiber bouton

Optical analysis of synaptic plasticity in rat hippocampus

Long-term potentiation (LTP) in the CA1 region of the hippocampus is dependent on NMDA receptor activation. Downstream of NMDA receptor signaling, the activation of α-calcium/calmodulin-dependent protein kinase II (αCaMKII) is both necessary and sufficient for the induction of this form of LTP. It has been shown that αCaMKII accumulates in spines after glutamate application or ‘chemical LTP’. This postsynaptic accumulation of αCaMKII could be a key step for the induction of LTP, because it localizes the activated kinase close to the substrates of synaptic potentiation. It is not clear, however, what the threshold, time course of αCaMKII translocation are, and whether it is specific to the stimulated synapses only. To address these three questions, I combined optical stimulation techniques (Channelrhodopsin-2 stimulation and two-photon glutamate uncaging) with optical measurements of calcium transients and αCaMKII concentration. This ‘all-optical’ approach made it possible to investigate synapse-specific changes during the induction of LTP. I could show that coincident activity of pre- and postsynaptic cells was needed to trigger the translocation of αCaMKII. Functional potentiation could be measured immediately after stimulation, whereas αCaMKII accumulation reached its peak ~10 min later. This points to an additional structural role of αCaMKII at the postsynaptic density. Both αCaMKII fractions, the cytoplasmic fraction and postsynaptic bound αCaMKII, increased after optical LTP induction. These changes were restricted to stimulated spines. In spines that showed a persistent volume increase, the amount of bound αCaMKII was increased by a factor of two after 30-40 minutes. A second very interesting finding was the close correlation between spine volume changes and LTP, in terms of the time course, induction threshold and specificity. The optical LTP protocol led to a lasting volume increase only in the stimulated spines, but not in directly neighboring spines on the same dendrite. Spine volume reached its maximum immediately after stimulation. Since my all-optical approach relied heavily on the use of a newly identified light-gated cation channel (Channelrhodopsin-2, ChR2), I finally also characterized light activation of ChR2 in hippocampal pyramidal cells in detail. Neuronal activity could be controlled by blue light with millisecond precision. No direct activation of ChR2 was observed by two-photon imaging lasers, making it possible to combine the ChR2 stimulation technique with two-photon imaging. This led to a third important finding: the release probability of ChR2-expressing axonal terminals was increased if the action potential was induced by light. As a result, pairing of light stimulation with postsynaptic depolarization induced reliable long-term potentiation at CA1 synapses. In summary, the new all-optical approach that combines ChR2 stimulation, two-photon glutamate uncaging, and optical measurements of calcium transients and protein concentration, provides a new avenue for investigating plasticity at the level of single synapses. The induction of LTP in single synapses revealed that accumulation of αCaMKII is input specific thus validating Hebb’s postulate on a micrometer scale

Markov chain models of calcium puffs and sparks

Localized cytosolic Ca2+ elevations known as puffs and sparks are important regulators of cellular function that arise due to the cooperative activity of Ca2+-regulated inositol 1,4,5-trisphosphate receptors (IP3Rs) or ryanodine receptors (RyRs) co-localized at Ca2+ release sites on the surface of the endoplasmic reticulum or sarcoplasmic reticulum. Theoretical studies have demonstrated that the cooperative gating of a cluster of Ca2+-regulated Ca 2+ channels modeled as a continuous-time discrete-state Markov chain may result in dynamics reminiscent of Ca2+ puffs and sparks. In such simulations, individual Ca2+-release channels are coupled via a mathematical representation of the local [Ca2+] and exhibit stochastic Ca2+ excitability where channels open and close in a concerted fashion. This dissertation uses Markov chain models of Ca 2+ release sites to advance our understanding of the biophysics connecting the microscopic parameters of IP3R and RyR gating to the collective phenomenon of puffs and sparks.;The dynamics of puffs and sparks exhibited by release site models that include both Ca2+ coupling and nearest-neighbor allosteric coupling are studied. Allosteric interactions are included in a manner that promotes the synchronous gating of channels by stabilizing neighboring closed-closed and/or open-open channel pairs. When the strength of Ca2+-mediated channel coupling is systematically varied, simulations that include allosteric interactions often exhibit more robust Ca2+ puffs and sparks. Interestingly, the changes in puff/spark duration, inter-event interval, and frequency observed upon the random removal of allosteric couplings that stabilize closed-closed channel pairs are qualitatively different than the changes observed when open-open channel pairs, or both open-open and closed-closed channel pairs are stabilized. The validity of a computationally efficient mean-field reduction applicable to the dynamics of a cluster of Ca2+-release Ca2+ channels coupled via the local [Ca2+] and allosteric interactions is also investigated.;Markov chain models of Ca2+ release sites composed of channels that are both activated and inactivated by Ca2+ are used to clarify the role of Ca2+ inactivation in the generation and termination of puffs and sparks. It is found that when the average fraction of inactivated channels is significant, puffs and sparks are often less sensitive to variations in the number of channels at release sites and the strength of Ca2+ coupling. While excessively fast Ca2+ inactivation can preclude puffs and sparks moderately fast Ca2+ inactivation often leads to time-irreversible puff/sparks whose termination is facilitated by the recruitment of inactivated channels throughout the duration of the puff/spark event. On the other hand, Ca2+ inactivation may be an important negative feedback mechanism even when its time constant is much greater than the duration of puffs and sparks. In fact, slow Ca 2+ inactivation can lead to release sites with a substantial fraction of inactivated channels that exhibit nearly time-reversible puffs and sparks that terminate without additional recruitment of inactivated channels

Calcium dynamics in dendrites and spines of spiny neurons in the somatosensory ‘barrel’ cortex of the rat

Two-photon excitation fluorescence microscopy was combined with the patch-clamp technique to study the Ca2+ dynamics in dendrites and spines of spiny neurons of layer 4 of the somatosensory cortex in acute thalamocortical brain slices of young (P13-P15) rats. Back-propagating action potentials (bAPs) resulted in a transient rise in Ca2+ in all dendrites and spines tested, representing a global intracellular chemical signal about the activity of the cell. In contrast, synaptically evoked excitatory postsynaptic potentials (EPSPs) resulted in a synapse specific, local increase in Ca2+. Pairing both stimuli at different inter-stimulus intervals revealed a precisely tuned coincidence detection mechanism for pre- and postsynaptic activity, coded in the peak Ca2+ transient amplitude. Linear, sub- and supralinear summation of the Ca2+ transients, depending on the time interval and the order of bAP and EPSP, was found. Ca2+ influx was maximal when the action potential followed synaptic stimulation within less than 20 ms. The mechanism of maximal Ca2+ influx could be explained by the properites of the NMDA receptor channel, which was activated by binding glutamate during synaptic stimulation and subsequent relief of the Mg2+ block by the bAP. Coincidence detection was restricted to the synaptic contact and it did not depend on the distance of the contact from the soma. This temporally and spatially highly restricted coincidence detection mechanism, which emplyed the Ca2+ transient amplitude as a readout signal might serve as an input specific trigger for spike-timing dependent plasticity. Indeed potentiation of EPSPs to 150% of the baseline amplitude could be induced by pairing extracellular stimulation with bAPs within the coincidence detection interval. Reversing the order of the stimuli resulted in depression of the EPSP amplitude to 70%. Thus it was concluded that spiny neurons in layer 4 of the juvenile rat barrel cortex exhibit spike-timing dependent plasticity, which corresponded well to the Ca2+ code used by their spines for coincidence detection

Spatiotemporal calcium-dynamics in presynaptic terminals

This thesis deals with a newly-developed model for the spatiotemporal calcium dynamics within presynaptic terminals. The model is based on single-protein kinetics and has been used to successfully describe different neuron types such as pyramidal neurons in the rat neocortex and the Calyx of Held of neurons from the rat brainstem. A limited number of parameters had to be adjusted to fluorescence measurements of the calcium concentration. These values can be interpreted as a prediction of the model, and in particular the protein densities can be compared to independent experiments. The contribution of single proteins to the total calcium dynamics has been analysed in detail for voltage-dependent calcium channel, plasma-membrane calcium ATPase, sodium-calcium exchanger, and endogenous as well as exogenous buffer proteins. The model can be used to reconstruct the unperturbed calcium dynamics from measurements using fluorescence indicators. The calcium response to different stimuli has been investigated in view of its relevance for synaptic plasticity. This work provides a first step towards a description of the complete synaptic transmission using single-protein data

Changes in zebra finch central nervous system morphology associated with developmental cannabinoid exposure

Adolescent CNS development is a highly organized, coordinated process that is both genetically and environmentally influenced, and is characterized by a period of dynamic, activity-dependent changes in synaptic connectivity. Growing evidence suggests that adolescent cannabis use is a risk factor for the development of persistent alterations in brain function. Taeniopygia guttata, the zebra finch, was used as a model of cannabinoid-altered vocal learning. We explored effects of cannabinoid-altered signaling during normal late-postnatal CNS development, as well as lasting morphological changes following exogenous cannabinoid exposure. In these studies, the cannabinoid agonist WIN 55,212-2 was administered to developing male zebra finches during sensorimotor song learning and dendritic spine densities measured following Golgi-Cox impregnation. Within HVC, a region necessary for songbird vocal production, and Area X, a striatal region essential for song learning, dendritic spines were inappropriately elevated by an average of 25% following developmental cannabinoid treatment. Treatments of adults that had already learned song were not associated with spine density changes. Cannabinoid-altered song and neuronal morphology were correlated with changes in levels of proteins related to cell signaling and morphology, including axonal Nf-200 and dendritic MAP2. After sensorimotor developmental cannabinoid treatment, anti-Nf-200 and -MAP2 antibodies were used to immunohistochemically confirm Golgi-Cox staining results. In the same brain areas where dendritic spines were elevated following CB agonist treatment, Nf-200 and MAP2 immunoreactivity (ir) were also elevated. To investigate mechanisms of cannabinoid-induced changes in neuronal morphology, we measured expression of the cytoskeletal protein Arc in NCM. A single exposure to novel song increased the postsynaptic densities of Arc protein. Two exposures to song were sufficient to produce habituation of this response. Habituation was prevented by pretreatment with WIN. These findings suggest there is a persistent, developmentally-restricted condition during periadolescence, and that cannabinoid agonism interferes with sensory integration and encoding necessary for accurate formation of memories.  Ph.D

Distribution and regulation of ion channels in neurons: Quantitative studies of global ion channel transport and homeostatic synaptic scaling

A healthy neuron must continually produce millions of proteins and distribute them to function-specific regions of the cell. Among these proteins are ion channels that modulate neuronal excitability, allowing neurons to fulfill their primary role of information transfer. Neurons are unique among cells in their morphology, with projections that extend hundreds to thousands of microns. Neuron size and asymmetry pose a challenge for autoregulation of properties that require cargo transport across the cell. Homeostasis of ion channel localization has strong implications for neural excitability. This thesis concerns the intracellular distribution of ion channels in the context of longitudinal transport and global neuron regulation. The principal contributions are experimental measurements, data analysis, and modeling in the study of longitudinal neurite transport. Empirical investigations focus on the distribution and trafficking kinetics of ion channel Kv4.2, including quantitative measurements of both passive diffusion and active microtubule-based transport in both axons and dendrites (Chapters 3 and 5). Mass action models reveal that measured transport profiles corroborate discrepancies in Kv4.2 localization both between neurite types and along the somatodendritic axis (Chapter 4). Exchange between mobile and immobile fractions, inferred from analysis of repeated photobleaching, shapes intracellular distribution of Kv4.2 (Chapter 5). Further, the ensuing theoretical study surveys global regulation of ion channels, specifically for synaptic scaling, which requires cell-wide modulation of AMPA receptors for normalization of neural excitability. A unified model of synaptic potentiation, transport, and feedback reveals limitations imposed on synaptic scaling by neuron morphology. A neuron balances the stability, accuracy, and efficiency of synaptic scaling (Chapter 6).National Institutes of Health Oxford-Cambridge Scholars Gates Cambridge University of North Carolina Medical Scientist Training Progra

Induction and Maintenance of Synaptic Plasticity

Synaptic long-term modifications following neuronal activation are believed to be at the origin of learning and long-term memory. Recent experiments suggest that these long-term synaptic changes are all-or-none switch-like events between discrete states of a single synapse. The biochemical network involving calcium/calmodulin-dependent protein kinase II (CaMKII) and its regulating protein signaling cascade has been hypothesized to durably maintain the synaptic state in form of a bistable switch. Furthermore, it has been shown experimentally that CaMKII and associated proteins such as protein kinase A and calcineurin are necessary for the induction of long-lasting increases (long-term potentiation, LTP) and/or long-lasting decreases (long-term depression, LTD) of synaptic efficacy. However, the biochemical mechanisms by which experimental LTP/LTD protocols lead to corresponding transitions between the two states in realistic models of such networks are still unknown. We present a detailed biochemical model of the calcium/calmodulin-dependent autophosphorylation of CaMKII and the protein signaling cascade governing the dephosphorylation of CaMKII. As previously shown, two stable states of the CaMKII phosphorylation level exist at resting intracellular calcium concentrations. Repetitive high calcium levels switch the system from a weakly- to a highly phosphorylated state (LTP). We show that the reverse transition (LTD) can be mediated by elevated phosphatase activity at intermediate calcium levels. It is shown that the CaMKII kinase-phosphatase system can qualitatively reproduce plasticity results in response to spike-timing dependent plasticity (STDP) and presynaptic stimulation protocols. A reduced model based on the CaMKII system is used to elucidate which parameters control the synaptic plasticity outcomes in response to STDP protocols, and in particular how the plasticity results depend on the differential activation of phosphatase and kinase pathways and the level of noise in the calcium transients. Our results show that the protein network including CaMKII can account for (i) induction - through LTP/LTD-like transitions - and (ii) storage - due to its bistability - of synaptic changes. The model allows to link biochemical properties of the synapse with phenomenological 'learning rules' used by theoreticians in neural network studies