Three-Dimensional Analysis of Spiny Dendrites Using Straightening and Unrolling Transforms

Current understanding of the synaptic organization of the brain depends to a large extent on knowledge about the synaptic inputs to the neurons. Indeed, the dendritic surfaces of pyramidal cells (the most common neuron in the cerebral cortex) are covered by thin protrusions named dendritic spines. These represent the targets of most excitatory synapses in the cerebral cortex and therefore, dendritic spines prove critical in learning, memory and cognition. This paper presents a new method that facilitates the analysis of the 3D structure of spine insertions in dendrites, providing insight on spine distribution patterns. This method is based both on the implementation of straightening and unrolling transformations to move the analysis process to a planar, unfolded arrangement, and on the design of DISPINE, an interactive environment that supports the visual analysis of 3D patterns.

Functional and effective connectivity in MEG. Application to the study of epilepsy

Nuestro cerebro contiene cerca de 1014 sinapsis neuronales. Esta enorme cantidad de conexiones proporciona un entorno ideal donde distintos grupos de neuronas se sincronizan transitoriamente para provocar la aparición de funciones cognitivas, como la percepción, el aprendizaje o el pensamiento. Comprender la organización de esta compleja red cerebral en base a datos neurofisiológicos, representa uno de los desafíos más importantes y emocionantes en el campo de la neurociencia. Se han propuesto recientemente varias medidas para evaluar cómo se comunican las diferentes partes del cerebro a diversas escalas (células individuales, columnas corticales, o áreas cerebrales). Podemos clasificarlos, según su simetría, en dos grupos: por una parte, la medidas simétricas, como la correlación, la coherencia o la sincronización de fase, que evalúan la conectividad funcional (FC); mientras que las medidas asimétricas, como la causalidad de Granger o transferencia de entropía, son capaces de detectar la dirección de la interacción, lo que denominamos conectividad efectiva (EC). En la neurociencia moderna ha aumentado el interés por el estudio de las redes funcionales cerebrales, en gran medida debido a la aparición de estos nuevos algoritmos que permiten analizar la interdependencia entre señales temporales, además de la emergente teoría de redes complejas y la introducción de técnicas novedosas, como la magnetoencefalografía (MEG), para registrar datos neurofisiológicos con gran resolución. Sin embargo, nos hallamos ante un campo novedoso que presenta aun varias cuestiones metodológicas sin resolver, algunas de las cuales trataran de abordarse en esta tesis. En primer lugar, el creciente número de aproximaciones para determinar la existencia de FC/EC entre dos o más señales temporales, junto con la complejidad matemática de las herramientas de análisis, hacen deseable organizarlas todas en un paquete software intuitivo y fácil de usar. Aquí presento HERMES (http://hermes.ctb.upm.es), una toolbox en MatlabR, diseñada precisamente con este fin. Creo que esta herramienta será de gran ayuda para todos aquellos investigadores que trabajen en el campo emergente del análisis de conectividad cerebral y supondrá un gran valor para la comunidad científica. La segunda cuestión practica que se aborda es el estudio de la sensibilidad a las fuentes cerebrales profundas a través de dos tipos de sensores MEG: gradiómetros planares y magnetómetros, esta aproximación además se combina con un enfoque metodológico, utilizando dos índices de sincronización de fase: phase locking value (PLV) y phase lag index (PLI), este ultimo menos sensible a efecto la conducción volumen. Por lo tanto, se compara su comportamiento al estudiar las redes cerebrales, obteniendo que magnetómetros y PLV presentan, respectivamente, redes más densamente conectadas que gradiómetros planares y PLI, por los valores artificiales que crea el problema de la conducción de volumen. Sin embargo, cuando se trata de caracterizar redes epilépticas, el PLV ofrece mejores resultados, debido a la gran dispersión de las redes obtenidas con PLI. El análisis de redes complejas ha proporcionado nuevos conceptos que mejoran caracterización de la interacción de sistemas dinámicos. Se considera que una red está compuesta por nodos, que simbolizan sistemas, cuyas interacciones se representan por enlaces, y su comportamiento y topología puede caracterizarse por un elevado número de medidas. Existe evidencia teórica y empírica de que muchas de ellas están fuertemente correlacionadas entre sí. Por lo tanto, se ha conseguido seleccionar un pequeño grupo que caracteriza eficazmente estas redes, y condensa la información redundante. Para el análisis de redes funcionales, la selección de un umbral adecuado para decidir si un determinado valor de conectividad de la matriz de FC es significativo y debe ser incluido para un análisis posterior, se convierte en un paso crucial. En esta tesis, se han obtenido resultados más precisos al utilizar un test de subrogadas, basado en los datos, para evaluar individualmente cada uno de los enlaces, que al establecer a priori un umbral fijo para la densidad de conexiones. Finalmente, todas estas cuestiones se han aplicado al estudio de la epilepsia, caso práctico en el que se analizan las redes funcionales MEG, en estado de reposo, de dos grupos de pacientes epilépticos (generalizada idiopática y focal frontal) en comparación con sujetos control sanos. La epilepsia es uno de los trastornos neurológicos más comunes, con más de 55 millones de afectados en el mundo. Esta enfermedad se caracteriza por la predisposición a generar ataques epilépticos de actividad neuronal anormal y excesiva o bien síncrona, y por tanto, es el escenario perfecto para este tipo de análisis al tiempo que presenta un gran interés tanto desde el punto de vista clínico como de investigación. Los resultados manifiestan alteraciones especificas en la conectividad y un cambio en la topología de las redes en cerebros epilépticos, desplazando la importancia del ‘foco’ a la ‘red’, enfoque que va adquiriendo relevancia en las investigaciones recientes sobre epilepsia. ABSTRACT There are about 1014 neuronal synapses in the human brain. This huge number of connections provides the substrate for neuronal ensembles to become transiently synchronized, producing the emergence of cognitive functions such as perception, learning or thinking. Understanding the complex brain network organization on the basis of neuroimaging data represents one of the most important and exciting challenges for systems neuroscience. Several measures have been recently proposed to evaluate at various scales (single cells, cortical columns, or brain areas) how the different parts of the brain communicate. We can classify them, according to their symmetry, into two groups: symmetric measures, such as correlation, coherence or phase synchronization indexes, evaluate functional connectivity (FC); and on the other hand, the asymmetric ones, such as Granger causality or transfer entropy, are able to detect effective connectivity (EC) revealing the direction of the interaction. In modern neurosciences, the interest in functional brain networks has increased strongly with the onset of new algorithms to study interdependence between time series, the advent of modern complex network theory and the introduction of powerful techniques to record neurophysiological data, such as magnetoencephalography (MEG). However, when analyzing neurophysiological data with this approach several questions arise. In this thesis, I intend to tackle some of the practical open problems in the field. First of all, the increase in the number of time series analysis algorithms to study brain FC/EC, along with their mathematical complexity, creates the necessity of arranging them into a single, unified toolbox that allow neuroscientists, neurophysiologists and researchers from related fields to easily access and make use of them. I developed such a toolbox for this aim, it is named HERMES (http://hermes.ctb.upm.es), and encompasses several of the most common indexes for the assessment of FC and EC running for MatlabR environment. I believe that this toolbox will be very helpful to all the researchers working in the emerging field of brain connectivity analysis and will entail a great value for the scientific community. The second important practical issue tackled in this thesis is the evaluation of the sensitivity to deep brain sources of two different MEG sensors: planar gradiometers and magnetometers, in combination with the related methodological approach, using two phase synchronization indexes: phase locking value (PLV) y phase lag index (PLI), the latter one being less sensitive to volume conduction effect. Thus, I compared their performance when studying brain networks, obtaining that magnetometer sensors and PLV presented higher artificial values as compared with planar gradiometers and PLI respectively. However, when it came to characterize epileptic networks it was the PLV which gives better results, as PLI FC networks where very sparse. Complex network analysis has provided new concepts which improved characterization of interacting dynamical systems. With this background, networks could be considered composed of nodes, symbolizing systems, whose interactions with each other are represented by edges. A growing number of network measures is been applied in network analysis. However, there is theoretical and empirical evidence that many of these indexes are strongly correlated with each other. Therefore, in this thesis I reduced them to a small set, which could more efficiently characterize networks. Within this framework, selecting an appropriate threshold to decide whether a certain connectivity value of the FC matrix is significant and should be included in the network analysis becomes a crucial step, in this thesis, I used the surrogate data tests to make an individual data-driven evaluation of each of the edges significance and confirmed more accurate results than when just setting to a fixed value the density of connections. All these methodologies were applied to the study of epilepsy, analysing resting state MEG functional networks, in two groups of epileptic patients (generalized and focal epilepsy) that were compared to matching control subjects. Epilepsy is one of the most common neurological disorders, with more than 55 million people affected worldwide, characterized by its predisposition to generate epileptic seizures of abnormal excessive or synchronous neuronal activity, and thus, this scenario and analysis, present a great interest from both the clinical and the research perspective. Results revealed specific disruptions in connectivity and network topology and evidenced that networks’ topology is changed in epileptic brains, supporting the shift from ‘focus’ to ‘networks’ which is gaining importance in modern epilepsy research.

Random positions of dendritic spines in human cerebral cortex

Dendritic spines establish most excitatory synapses in the brain and are located in Purkinje cell’s dendrites along helical paths, perhaps maximizing the probability to contact different axons. To test whether spine helixes also occur in neocortex, we reconstructed >500 dendritic segments from adult human cortex obtained from autopsies. With Fourier analysis and spatial statistics, we analyzed spine position along apical and basal dendrites of layer 3 pyramidal neurons from frontal, temporal, and cingulate cortex. Although we occasionally detected helical positioning, for the great majority of dendrites we could not reject the null hypothesis of spatial randomness in spine locations, either in apical or basal dendrites, in neurons of different cortical areas or among spines of different volumes and lengths. We conclude that in adult human neocortex spine positions are mostly random. We discuss the relevance of these results for spine formation and plasticity and their functional impact for cortical circuits.

Random positioning of dendritic spines in the human cerebral cortex

Dendritic spines establish most excitatory synapses in the brain and are located in Purkinje cell?s dendrites along helical paths, perhaps maximizing the probability to contact different axons. To test whether spine helixes also occur in neocortex, we reconstructed ?500 dendritic segments from adult human cortex obtained from autopsies. With Fourier analysis and spatial statistics, we analyzed spine position along apical and basal dendrites of layer 3 pyramidal neurons from frontal, temporal, and cingulate cortex. Although we occasionally detected helical positioning, for the great majority of dendrites we could not reject the null hypothesis of spatial randomness in spine locations, either in apical or basal dendrites, in neurons of different cortical areas or among spines of different volumes and lengths. We conclude that in adult human neocortex spine positions are mostly random. We discuss the relevance of these results for spine formation and plasticity and their functional impact for cortical circuits.

Evidence of a role for cyclic ADP-ribose in long-term synaptic depression in hippocampus

Ca2+ released from presynaptic and postsynaptic intracellular stores plays important roles in activity-dependent synaptic plasticity, including long-term depression (LTD) of synaptic strength. At Schaffer collateral–CA1 synapses in the hippocampus, presynaptic ryanodine receptor-gated stores appear to mobilize some of the Ca2+ necessary to induce LTD. Cyclic ADP-ribose (cADPR) has recently been proposed as an endogenous activator of ryanodine receptors in sea urchin eggs and several mammalian cell types. Here, we provide evidence that cADPR-mediated signaling pathways play a key role in inducing LTD. We show that biochemical production of cGMP increases cADPR concentration in hippocampal slices in vitro, and that blockade of cGMP-dependent protein kinase, cADPR receptors, or ryanodine-sensitive Ca2+ stores each prevent the induction of LTD at Schaffer collateral–CA1 synapses. A lack of effect of postsynaptic infusion of either cADPR antagonist indicates a probable presynaptic site of action.

A nerve growth factor mimetic TrkA antagonist causes withdrawal of cortical cholinergic boutons in the adult rat

Cholinergic neurons respond to the administration of nerve growth factor (NGF) in vivo with a prominent and selective increase of choline acetyl transferase activity. This suggests the possible involvement of endogenous NGF, acting through its receptor TrkA, in the maintenance of central nervous system cholinergic synapses in the adult rat brain. To test this hypothesis, a small peptide, C(92-96), that blocks NGF-TrkA interactions was delivered stereotactically into the rat cortex over a 2-week period, and its effect and potency were compared with those of an anti-NGF monoclonal antibody (mAb NGF30). Two presynaptic antigenic sites were studied by immunoreactivity, and the number of presynaptic sites was counted by using an image analysis system. Synaptophysin was used as a marker for overall cortical synapses, and the vesicular acetylcholine transporter was used as a marker for cortical cholinergic presynaptic sites. No significant variations in the number of synaptophysin-immunoreactive sites were observed. However, both mAb NGF30 and the TrkA antagonist C(92-96) provoked a significant decrease in the number and size of vesicular acetylcholine transporter–IR sites, with the losses being more marked in the C(92-96) treated rats. These observations support the notion that endogenously produced NGF acting through TrkA receptors is involved in the maintenance of the cholinergic phenotype in the normal, adult rat brain and supports the idea that NGF normally plays a role in the continual remodeling of neural circuits during adulthood. The development of neurotrophin mimetics with antagonistic and eventually agonist action may contribute to therapeutic strategies for central nervous system degeneration and trauma.

Long-range signaling within growing neurites mediated by neurotrophin-3

In addition to well established trophic functions, neurotrophins acutely affect neurotransmitter secretion from the presynaptic nerve terminal, influence synaptic development, and may serve as selective retrograde messengers that regulate synaptic efficacy. The crucial question related to the mechanisms of neurotrophin-mediated signaling is whether acute effects of neurotrophins are spatially restricted to the activated synapses. Here we have used a local perfusion technique for local delivery of neurotrophin-3 (NT-3) to various regions of developing Xenopus embryo neurons in culture. Within minutes after a focal exposure of a soma or a small (≈30 μm in length) axonal segment to NT-3, we observed an increase in the spontaneous neurotransmitter secretion from the presynaptic nerve terminals located ≈300–400 μm away from the site of NT-3 application. Secretory activity along the axonal shaft was not affected. Our findings suggest that the NT-3-mediated signal may rapidly travel through neuronal cytoplasm over unexpectedly long distances and modulate neurotransmitter release specifically at the presynaptic nerve terminals.

In vivo evidence that erythropoietin protects neurons from ischemic damage

Erythropoietin (EPO) produced by the kidney and the liver (in fetuses) stimulates erythropoiesis. In the central nervous system, neurons express EPO receptor (EPOR) and astrocytes produce EPO. EPO has been shown to protect primary cultured neurons from N-methyl-d-aspartate (NMDA) receptor-mediated glutamate toxicity. Here we report in vivo evidence that EPO protects neurons against ischemia-induced cell death. Infusion of EPO into the lateral ventricles of gerbils prevented ischemia-induced learning disability and rescued hippocampal CA1 neurons from lethal ischemic damage. The neuroprotective action of exogenous EPO was also confirmed by counting synapses in the hippocampal CA1 region. Infusion of soluble EPOR (an extracellular domain capable of binding with the ligand) into animals given a mild ischemic treatment that did not produce neuronal damage, caused neuronal degeneration and impaired learning ability, whereas infusion of the heat-denatured soluble EPOR was not detrimental, demonstrating that the endogenous brain EPO is crucial for neuronal survival. The presence of EPO in neuron cultures did not repress a NMDA receptor-mediated increase in intracellular Ca2+, but rescued the neurons from NO-induced death. Taken together EPO may exert its neuroprotective effect by reducing the NO-mediated formation of free radicals or antagonizing their toxicity.

Long-term potentiation and dual-component quantal signaling in the dentate gyrus

Long-term potentiation (LTP) of excitatory transmission is an important candidate cellular mechanism for the storage of memories in the mammalian brain. The subcellular phenomena that underlie the persistent increase in synaptic strength, however, are incompletely understood. A potentially powerful method to detect a presynaptic increase in glutamate release is to examine the effect of LTP induction on the rate at which the use-dependent blocker MK-801 attenuates successive N-methyl-d-aspartic acid (NMDA) receptor-mediated synaptic signals. This method, however, has given apparently contradictory results when applied in hippocampal CA1. The inconsistency could be explained if NMDA receptors were opened by glutamate not only released from local presynaptic terminals, but also diffusing from synapses on neighboring cells where LTP was not induced. Here we examine the effect of pairing-induced LTP on the MK-801 blocking rate in two afferent inputs to dentate granule cells. LTP in the medial perforant path is associated with a significant increase in the MK-801 blocking rate, implying a presynaptic increase in glutamate release probability. An enhanced MK-801 blocking rate is not seen, however, in the lateral perforant path. This result still could be compatible with a presynaptic contribution to LTP in the lateral perforant path if intersynaptic cross-talk occurred. In support of this hypothesis, we show that NMDA receptors consistently sense more quanta of glutamate than do α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors. In the medial perforant path, in contrast, there is no significant difference in the number of quanta mediated by the two receptors. These results support a presynaptic contribution to LTP and imply that differences in intersynaptic cross-talk can complicate the interpretation of experiments designed to detect changes in transmitter release.

R-type Ca2+ currents evoke transmitter release at a rat central synapse

Voltage-dependent Ca2+ currents evoke synaptic transmitter release. Of six types of Ca2+ channels, L-, N-, P-, Q-, R-, and T-type, only N- and P/Q-type channels have been pharmacologically identified to mediate action-potential-evoked transmitter release in the mammalian central nervous system. We tested whether Ca2+ channels other than N- and P/Q-type control transmitter release in a calyx-type synapse of the rat medial nucleus of the trapezoid body. Simultaneous recordings of presynaptic Ca2+ influx and the excitatory postsynaptic current evoked by a single action potential were made at single synapses. The R-type channel, a high-voltage-activated Ca2+ channel resistant to L-, N-, and P/Q-type channel blockers, contributed 26% of the total Ca2+ influx during a presynaptic action potential. This Ca2+ current evoked transmitter release sufficiently large to initiate an action potential in the postsynaptic neuron. The R-type current controlled release with a lower efficacy than other types of Ca2+ currents. Activation of metabotropic glutamate receptors and γ-aminobutyric acid type B receptors inhibited the R-type current. Because a significant fraction of presynaptic Ca2+ channels remains unidentified in many other central synapses, the R-type current also could contribute to evoked transmitter release in these synapses.

Dynamic mapping at the laminar level of odor-elicited responses in rat olfactory bulb by functional MRI

We have applied functional MRI (fMRI) based on blood oxygenation level-dependent (BOLD) image-contrast to map odor-elicited olfactory responses at the laminar level in the rat olfactory bulb (OB) elicited by iso-amyl acetate (10−2 dilution of saturated vapor) with spatial and temporal resolutions of 220×220×1,000 μm and 36 s. The laminar structure of the OB was clearly depicted by high-resolution in vivo anatomical MRI with spatial resolution of 110×110×1,000 μm. In repeated BOLD fMRI measurements, highly significant (P < 0.001) foci were located in the outer layers of both OBs. The occurrence of focal OB activity within a domain at the level of individual glomeruli or groups of glomeruli was corroborated on an intra- and inter-animal basis under anesthetized conditions with this noninvasive method. The dynamic studies demonstrated that the odor-elicited BOLD activations were highly reproducible on a time scale of minutes, whereas over tens of minutes the activations sometimes varied slowly. We found large BOLD signal (ΔS/S = 10–30%) arising from the olfactory nerve layer, which is devoid of synapses and composed of unmyelinated fibers and glial cells. Our results support previous studies with other methods showing that odors elicit activity within glomerular layer domains in the mammalian OB, and extend the analysis to shorter time periods at the level of individual glomeruli or groups of glomeruli. With further improvement, BOLD fMRI should be ideal for systematic analysis of the functional significance of individual glomeruli in olfactory information encoding and of spatiotemporal processing within the olfactory system.

Activity differentially regulates the surface expression of synaptic AMPA and NMDA glutamate receptors

Distinct subtypes of glutamate receptors often are colocalized at individual excitatory synapses in the mammalian brain yet appear to subserve distinct functions. To address whether neuronal activity may differentially regulate the surface expression at synapses of two specific subtypes of ionotropic glutamate receptors we epitope-tagged an AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) receptor subunit (GluR1) and an NMDA (N-methyl-d-aspartate) receptor subunit (NR1) on their extracellular termini and expressed these proteins in cultured hippocampal neurons using recombinant adenoviruses. Both receptor subtypes were appropriately targeted to the synaptic plasma membrane as defined by colocalization with the synaptic vesicle protein synaptophysin. Increasing activity in the network of cultured cells by prolonged blockade of inhibitory synapses with the γ-aminobutyric acid type A receptor antagonist picrotoxin caused an activity-dependent and NMDA receptor-dependent decrease in surface expression of GluR1, but not NR1, at synapses. Consistent with this observation identical treatment of noninfected cultures decreased the contribution of endogenous AMPA receptors to synaptic currents relative to endogenous NMDA receptors. These results indicate that neuronal activity can differentially regulate the surface expression of AMPA and NMDA receptors at individual synapses.

Differential roles of Ca2+/calmodulin-dependent kinases in posttetanic potentiation at input selective glutamatergic pathways

The electrosensory lateral line lobe (ELL) of the electric fish Apteronotus leptorhynchus is a layered medullary region receiving electroreceptor input that terminates on basal dendrites of interneurons and projection (pyramidal) cells. The molecular layer of the ELL contains two distinct glutamatergic feedback pathways that terminate on the proximal (ventral molecular layer, VML) and distal (dorsal molecular layer) apical dendrites of pyramidal cells. Western blot analysis with an antibody directed against mammalian Ca2+/calmodulin-dependent kinase 2, α subunit (CaMK2α) recognized a protein of identical size in the brain of A. leptorhynchus. Immunohistochemistry demonstrated that CaMK2 α expression in the ELL was restricted to fibers and terminals in the VML. Posttetanic potentiation (PTP) could be readily elicited in pyramidal cells by stimulation of either VML or DML in brain slices of the ELL. PTP in the VML was blocked by extracellular application of a CaMK2 antagonist (KN62) while intracellular application of KN62 or a CaMK2 inhibitory peptide had no effect, consistent with the presynaptic localization of CaMK2 α in VML. PTP in the dorsal molecular layer was not affected by extracellular application of KN62. Anti-Hebbian plasticity has also been demonstrated in the VML, but was not affected by KN62. These results demonstrate that, while PTP can occur independent of CaMK2, it is, in some synapses, dependent on this kinase.

Omega images at the active zone may be endocytotic rather than exocytotic: Implications for the vesicle hypothesis of transmitter release

A Ca2+-dependent synaptic vesicle-recycling pathway emanating from the plasma membrane adjacent to the dense body at the active zone has been demonstrated by blocking pinch-off of recycling membrane by using the Drosophila mutant, shibire. Exposure of wild-type Drosophila synapses to low Ca2+/high Mg2+ saline is shown here to block this active zone recycling pathway at the stage in which invaginations of the plasma membrane develop adjacent to the dense body. These observations, in combination with our previous demonstration that exposure to high Ca2+ causes “docked” vesicles to accumulate in the identical location where active zone endocytosis occurs, suggest the possibility that a vesicle-recycling pathway emanating from the active zone may exist that is stimulated by exposure to elevated Ca2+, thereby causing an increase in vesicle recycling, and is suppressed by exposure to low Ca2+ saline, thereby blocking newly forming vesicles at the invagination stage. The presence of a Ca2+-dependent endocytotic pathway at the active zone opens up the following possibilities: (i) electron microscopic omega-shaped images (and their equivalent, freeze fracture dimples) observed at the active zone adjacent to the dense body could represent endocytotic images (newly forming vesicles) rather than exocytotic images; (ii) vesicles observed attached to the plasma membrane adjacent to the dense body could represent newly formed vesicles rather than vesicles “docked” for release of transmitter.

Activity-dependent regulation of synaptic clustering in a hippocampal culture system

Currently, there is a limited understanding of the factors that influence the localization and density of individual synapses in the central nervous system. Here we have studied the effects of activity on synapse formation between hippocampal dentate granule cells and CA3 pyramidal neurons in culture, taking advantage of FM1–43 as a fluorescent marker of synaptic boutons. We observed an early tendency for synapses to group together, quickly followed by the appearance of synaptic clusters on dendritic processes. These events were strongly influenced by N-methyl-d-aspartic acid receptor- and cyclic AMP-dependent signaling. The microstructure and localization of the synaptic clusters resembled that found in hippocampus, at mossy fiber synapses of stratum lucidum. Activity-dependent clustering of synapses represents a means for synaptic targeting that might contribute to synaptic organization in the brain.