Modeling the Contributions of the Exocytotic Machinery and Receptor Desensitization to Short- and Long-Term Plasticity of Synapses Between Neocortical Pyramidal Neurons

Short-term synaptic depression (STD) refers to the progressive decrease in synaptic efficacy during a spike train. This decrease may be explained in terms of presynaptic and postsynaptic processes, such as a decrease in the probability of transmitter release, and postsynaptic receptor desensitization. STD may be very strong, and is release-dependent in neocortical pyramid-pyramid synapses. Using a stochastic synapse model, we suggest that the main source of depression in these synapses is the step of vesicle priming, while vesicle depletion and postsynaptic receptor desensitization are proposed to play a lesser role. Our results suggest that vesicle priming may explain not only the release-dependent nature of STD, but also the observation that an average of about one vesicle per active zone is released in central synapses, without positing forced univesicular release. We propose that the latter phenomenon is due to a low priming probability. Our results also explain the effect of paired pre- and postsynaptic activity on STD. In neocortical pyramid-pyramid synapses pairing induces a form of long-term potentiation that has been described as a redistribution of synaptic efficacy (RSE). We propose that RSE is due to a pairing-induced increase in the probability that a primed vesicle will undergo release in response to a presynaptic action potential. This increase may be due to an increased Ca^2+ influx through voltage-gated Ca^2+ channels, or to an increased sensitivity of primed vesicles to this influx. The results were obtained by constraining the model with experimentally observed levels of release probability and other synaptic variables.Defense Advanced Research Projects Agency and the Office of Naval Research (N00014-95-l-0409); Office of Naval Research (N00014-95-l-0657)

Cellular specializations for sound localization

One of the key elements in auditory perception is the localization of sounds in space. The major cues used for localizing sounds in the azimuthal plane have long been recognized as interaural differences in time of arrival of a sound and amplitude differences between the two ears (Rayleigh 1907; Thompson 1878). High frequency sounds are reflected by the head and thereby produce interaural level differences (ILDs) that are used for localization. The head does not reflect low frequency sounds and so interaural timing differences (ITDs) are used. One of the cell groups of the auditory brainstem, the medial superior olive (MSO), functions in sound localization by comparing ITDs between the two ears. The MSO is defined as a binaural group of cells because it integrates input from the cochlear nucleus (CN) from each ear. Afferent nerve fibers from the ipsilateral CN are restricted to dendrites oriented laterally and inputs from the contralateral CN are segregated to medially oriented dendrites (Stotler 1953). At low to moderate sound levels, activation from each cochlear nucleus is below action potential threshold and MSO neurons only generate action potentials when inputs from both sides arrive within a short temporal window called the coincidence detection window.;Several cellular specializations exist along the auditory pathway that aid MSO cells in their ability to detect changes in ITD. These specializations include large nerve terminals and distinct organelle complexes located within terminals, which facilitate fast, well-timed inhibitory inputs to MSO cells. Very little is known about the role of inhibition in sound localization and proper understanding of its role depends on knowledge of the cells that impinge on the MSO and the pharmacology and kinetics of synaptic transmission in MSO cells. Also, the membranes of MSO cells contain specific voltage-gated potassium channels (Kv), these channels are known to affect membrane electrical properties, but how these channels influence ITD sensitivity is unknown. The main goal of my research was to understand these cellular specializations that contribute to neural processing of ITDs

Synaptic activity and the formation and maintenance of neuronal circuits

One of the most fundamental features of neurons is their polarized organization with two types of neurites extending from the cell body, axons and dendrites that are both functionally and morphologically distinct. During development, both axons and dendrites possess highly dynamic and actin-rich growth cones and filopodia extending from their shafts, which are subsequently replace by fundamentally stable axonal varicosities and dendritic spines. Together they form the basic elements of mature synapses. To mimic in vivo neuronal development, I have used organotypic cultures of brain tissue from transgenic mice expressing either green fluorescent protein (GFP) bearing a surface membrane localization signal or actin-GFP in combination with live cell imaging system. This approach provided me with high-resolution images of developing neurons’ fine structure in organized tissue. Co-cultures of fluorescent and non-fluorescent hippocampal slices enabled me then to examine simultaneously dendrite differentiation in the fluorescent slice and to track the fate of fluorescent axons growing into the non-fluorescent slice. Together this granted me a powerful tool to study neuronal network formation and developmental maturation of axons and dendrites. Co-cultures of embryonic tissue showed a sustained cross-innervation of axonal projections. Over time neurons in these co-cultures formed a dense axonal network with numerous axonal varicosities along their shaft. This axonal plexus remained present beyond 2 months in vitro. Dendrites in these embryonic co-cultures subsequently switched from producing labile filopodia to fundamentally stable dendritic spines. These mature dendritic spines had morphologies similar to those reported from studies of adult brain. Both axons and dendrites exhibited a successive focalisation of actin-based dynamics to the site of the synaptic junction. The observed changes in shape of mature axonal varicosities and dendritic spines together with the rapidly extension and retraction of actin-rich protrusions from the top of varicosities and spine heads suggest a retained capacity for experience-dependent fine-tuning e.g. during either periods of learning and memory or during brain damage resulting in an altered connectivity for both pre- and postsynaptic compartments in the mature mammalian central nervous system. The observed morphological dynamics suggest a high degree of preservation of morphological plasticity at the synapse in mature neuronal networks. Co-cultures of postnatal brain slices showed intensive invasion of axonal projections during the first two weeks in culture, followed by dramatic axonal regression and resulting in a near complete absence of cross-innervating axons after 1 month in vitro. In contrast, dendrite development in each of these postnatal cultures was fundamentally normal and occurred similar to that observed in embryonic co-cultures. I then co-cultured embryonic and postnatal slices to investigate whether the difference in capacity to cross-innervate between postnatal co-cultures and embryonic co-cultures were the result of tissue maturation. We found that the postnatal slice degenerated so that after 1 month in culture it had almost disappeared whereas the neighbouring embryonic slice had matured without noticeable problems. Staining these co-cultures of embryonic and postnatal slices showed a massive invasion of microglial cells into the dying postnatal slice. The difference between embryonic and postnatal neurons in their capacity to maintain cross-innervating synaptic connection suggests the existence of a developmental switch resulting in the inability of sustained afferent cross-innervation between postnatal brain slices. At the same time, in heterochronic co-cultures it causes miscommunication between postnatal and embryonic cells leading to profound degeneration of postnatal tissue. The thick layer of microglia surrounding postnatal tissue suggests their involvement in neuronal degeneration similar to that observed in axotomy-induced neuronal death and various neurodegenerative conditions such as Alzheimer’s disease. The earlier suggested preservation of morphological plasticity at the synapse in mature neuronal networks was illustrated by cooling mature hippocampal slices, either acutely cut brain slices or organotypic cultures, to room temperature. Dendritic spines are highly sensitive to reduced temperature with rapid loss of actin-based motility followed by disappearance of the entire spine structure within 12 hours. However, rewarming these cooled slices to 37˚C resulted in the rapid extension of filopodia from the surface of dendrites and re-establishment of dendritic spines within several of hours. These data underline the high degree of plasticity retained by neuronal connections in the mature CNS and suggest a link between dendritic spine structure and global brain function

Estimating the glutamate transporter surface density in distinct sub-cellular compartments of mouse hippocampal astrocytes

Glutamate transporters preserve the spatial specificity of synaptic transmission by limiting glutamate diffusion away from the synaptic cleft, and prevent excitotoxicity by keeping the extracellular concentration of glutamate at low nanomolar levels. Glutamate transporters are abundantly expressed in astrocytes, and previous estimates have been obtained about their surface expression in astrocytes of the rat hippocampus and cerebellum. Analogous estimates for the mouse hippocampus are currently not available. In this work, we derive the surface density of astrocytic glutamate transporters in mice of different ages via quantitative dot blot. We find that the surface density of glial glutamate transporters is similar in 7-8 week old mice and rats. In mice, the levels of glutamate transporters increase until about 6 months of age and then begin to decline slowly. Our data, obtained from a combination of experimental and modeling approaches, point to the existence of stark differences in the density of expression of glutamate transporters across different sub-cellular compartments, indicating that the extent to which astrocytes limit extrasynaptic glutamate diffusion depends not only on their level of synaptic coverage, but also on the identity of the astrocyte compartment in contact with the synapse. Together, these findings provide information on how heterogeneity in the spatial distribution of glutamate transporters in the plasma membrane of hippocampal astrocytes my alter glutamate receptor activation out of the synaptic cleft

Analog Spiking Neuromorphic Circuits and Systems for Brain- and Nanotechnology-Inspired Cognitive Computing

Human society is now facing grand challenges to satisfy the growing demand for computing power, at the same time, sustain energy consumption. By the end of CMOS technology scaling, innovations are required to tackle the challenges in a radically different way. Inspired by the emerging understanding of the computing occurring in a brain and nanotechnology-enabled biological plausible synaptic plasticity, neuromorphic computing architectures are being investigated. Such a neuromorphic chip that combines CMOS analog spiking neurons and nanoscale resistive random-access memory (RRAM) using as electronics synapses can provide massive neural network parallelism, high density and online learning capability, and hence, paves the path towards a promising solution to future energy-efficient real-time computing systems. However, existing silicon neuron approaches are designed to faithfully reproduce biological neuron dynamics, and hence they are incompatible with the RRAM synapses, or require extensive peripheral circuitry to modulate a synapse, and are thus deficient in learning capability. As a result, they eliminate most of the density advantages gained by the adoption of nanoscale devices, and fail to realize a functional computing system. This dissertation describes novel hardware architectures and neuron circuit designs that synergistically assemble the fundamental and significant elements for brain-inspired computing. Versatile CMOS spiking neurons that combine integrate-and-fire, passive dense RRAM synapses drive capability, dynamic biasing for adaptive power consumption, in situ spike-timing dependent plasticity (STDP) and competitive learning in compact integrated circuit modules are presented. Real-world pattern learning and recognition tasks using the proposed architecture were demonstrated with circuit-level simulations. A test chip was implemented and fabricated to verify the proposed CMOS neuron and hardware architecture, and the subsequent chip measurement results successfully proved the idea. The work described in this dissertation realizes a key building block for large-scale integration of spiking neural network hardware, and then, serves as a step-stone for the building of next-generation energy-efficient brain-inspired cognitive computing systems

Developmental Changes in the Structure and Composition of the Postsynaptic Density

The development of the brain and its underlying circuitry is dependent on the formation of trillions of chemical synapses, which are highly specialized contacts that regulate the flow of information from one neuron to the next. It is through these synaptic connections that neurons wire together into networks capable of performing specific tasks, and activity-dependent changes in their structural and physiological state is one way that the brain is thought to adapt and store information. At the ultrastructural level, developmental and activity-dependent changes in the size and shape of dendritic spines have been well documented, and it is widely believed that structural changes in spines are a hallmark sign of synapse maturation and alteration of synaptic physiology. While changes in spine structure have been studied extensively, changes in one of its most prominent components, the postsynaptic density (PSD), have largely evaded observation. The PSD is a protein-rich organelle on the cytoplasmic side of the postsynaptic membrane, where it sits in direct opposition to the presynaptic terminal. The PSD functions both to cluster neurotransmitter receptors at the cell surface as well as organize the intracellular signaling molecules responsible for transducing extracellular signals to the postsynaptic cell. Much is known about the chemical composition of the PSD, but the structural arrangement of its molecular components is not well documented. Adding to the difficulty of understanding such a complex mass of protein machinery is the fact that its protein composition is known to change in response to synaptic activity, meaning that its structure is plastic and no two PSDs are identical. Here, immuno-gold labeling and electron tomography of PSDs isolated throughout development was used to track changes in both the structure and molecular composition of the PSD. State-of-the-art cryo-electron tomography was used to study the fine structure of the PSD during development, and provides an unprecedented glimpse into its molecular architecture in an un-fixed, unstained and hydrated state. Through this analysis, large structural and compositional changes are apparent and suggest a model by which the PSD is first assembled as a mesh-like lattice of proteins that function as support for the later recruitment of various PSD components. Spatial analysis of the recruitment of proteins into the PSD demonstrated that its assembly has an underlying order

Transfer Properties of the Hair Cell-Afferent Fiber Synapse

The perception of sound is initiated in the inner ear by the conversion of vibrational energy into a neural code, a transduction process achieved by the chemical synapses of hair cells in the auditory periphery. Thus, the operation of the hair cell’s presynaptic active zone is key to understanding auditory transduction. However, the lack of suitable experimental systems in which to investigate both the presynaptic and postsynaptic aspects of this synapse with high resolution has limited our understanding of its functional characteristics. This work describes the development of a novel in vitro preparation of the amphibian papilla from Rana catesbeiana that provides electrical access to the pre- and postsynaptic elements of the hair cell’s afferent synapse. The transfer properties of this ribbon-type synapse have been explored with a variety of electrophysiological techniques, including whole-cell recordings, capacitance measurements, and iontophoresis. Glutamate is released from hair cells in response to Ca2+ influx through L-type Ca2+ channels and is detected by AMPA receptors in postsynaptic fibers. Gradations in the extent of presynaptic stimulation are encoded by a linear increase in the postsynaptic response with respect to the presynaptic Ca2+ current, a relation imparted primarily by an increase in the frequency of release events. Both spontaneous and evoked postsynaptic signals are stereotyped in waveform but highly variable in amplitude. Determination of the size of the quantal response provides compelling evidence that the majority of these events are multiquantal. Multiquantal events may originate from individual active zones and do not typically saturate postsynaptic receptors, thus suggesting that they may have functional significance. The results presented in this study are most consistent with compound exocytosis as the dominant form of transmitter release at individual hair-cell active zones

Microiontophoresis as a technique to investigate Spike Timing Dependent Plasticity

Spike timing dependent plasticity (STDP) is a form of synaptic plasticity that depends on the relative time of activation of a presynaptic neuron and its postsynaptic neuron. STDP in the synapses made by Schaffer collateral afferents onto hippocampal CA1 pyramidal neurons (CA3-CA1 synapses) is NMDA receptor dependent. The objective of the current study was to develop and test a technique of glutamate iontophoresis that could replace the role of presynaptic neurotransmitter release at the CA3-CA1 synapse, so that the postsynaptic mechanisms involved in the induction of STDP could be isolated for study. Therefore, this document describes: (1) fabrication of electrodes that could be used for millisecond-level microiontophoresis in acute slice preparations of the juvenile rat hippocampus; (2) characterization of the properties and limitations of microiontophoresis in slice tissue, specifically for activation of postsynaptic ionotropic glutamate receptors at the CA3-CA1 synapse; (3) induction of STDP by pairing microiontophoresis with postsynaptic depolarization; (4) characterization of the properties and limitations of microiontophoretically induced STDP. It was determined that microiontophoresis is a viable technique to study the postsynaptic mechanisms of STDP at the CA3-CA1 synapse. My results also show that microiontophoretically induced STDP exhibits many of the same general properties as STDP induced either synaptically or by exogenously applied agonists. Microiontophoretically induced STDP also exhibits other features that will need to be considered during the design and interpretation of further experiments

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