Gotowa bibliografia na temat „Hippocampal Pyramidal Neuronal Dendrites”

In hippocampal pyramidal neurons, voltage-gated Ca2+ channels open in response to action potentials. This results in elevations in the intracellular concentration of Ca2+ that are maximal in the proximal apical dendrites and decrease rapidly with distance from the soma. The control of these action potential-evoked Ca2+ elevations is critical for the regulation of hippocampal neuronal activity. As part of Ca2+ signaling microdomains, small-conductance Ca2+-activated K+ (SK) channels have been shown to modulate the amplitude and duration of intracellular Ca2+ signals by feedback regulation of synaptically activated Ca2+ sources in small distal dendrites and dendritic spines, thus affecting synaptic plasticity in the hippocampus. In this study, we investigated the effect of the activation of SK channels on Ca2+ transients specifically induced by action potentials in the proximal processes of hippocampal pyramidal neurons. Our results, obtained by using selective SK channel blockers and enhancers, show that SK channels act in a feedback loop, in which their activation by Ca2+ entering mainly through L-type voltage-gated Ca2+ channels leads to a reduction in the subsequent dendritic influx of Ca2+. This underscores a new role of SK channels in the proximal apical dendrite of hippocampal pyramidal neurons.

Collapsin response mediator proteins (CRMPs) are highly expressed in the brain during early postnatal development and continue to be present in specific regions into adulthood, especially in areas with extensive neuronal plasticity including the hippocampus. They are found in the axons and dendrites of neurons wherein they contribute to specific signaling mechanisms involved in the regulation of axonal and dendritic development/maintenance. We previously identified CRMP3’s role on the morphology of hippocampal CA1 pyramidal dendrites and hippocampus-dependent functions. Our focus here was to further analyze its role in the dentate gyrus where it is highly expressed during development and in adults. On the basis of our new findings, it appears that CRMP3 has critical roles both in axonal and dendritic morphogenesis of dentate granular neurons. In CRMP3-deficient mice, the dendrites become dystrophic while the infrapyramidal bundle of the mossy fiber shows aberrant extension into the stratum oriens of CA3. This axonal misguided projection of granular neurons suggests that the mossy fiber-CA3 synaptic transmission, important for the evoked propagation of the activity of the hippocampal trisynaptic circuitry, may be altered, whereas the dystrophic dendrites may impair the dynamic interactions with the entorhinal cortex, both expected to affect hippocampal function.

Stability of neuronal connectivity is critical for brain functions, and morphological perturbations are associated with neurodegenerative disorders. However, how neuronal morphology is maintained in the adult brain remains poorly understood. Here, we identify Wnt5a, a member of the Wnt family of secreted morphogens, as an essential factor in maintaining dendritic architecture in the adult hippocampus and for related cognitive functions in mice. Wnt5a expression in hippocampal neurons begins postnatally, and its deletion attenuated CaMKII and Rac1 activity, reduced GluN1 glutamate receptor expression, and impaired synaptic plasticity and spatial learning and memory in 3-mo-old mice. With increased age, Wnt5a loss caused progressive attrition of dendrite arbors and spines in Cornu Ammonis (CA)1 pyramidal neurons and exacerbated behavioral defects. Wnt5a functions cell-autonomously to maintain CA1 dendrites, and exogenous Wnt5a expression corrected structural anomalies even at late-adult stages. These findings reveal a maintenance factor in the adult brain, and highlight a trophic pathway that can be targeted to ameliorate dendrite loss in pathological conditions.

The ability to trigger neuronal spiking activity is one of the most important functional characteristics of synaptic inputs and can be quantified as a measure of synaptic efficacy (SE). Using model neurons with both highly simplified and real morphological structures (from a single cylindrical dendrite to a hippocampal granule cell, CA1 pyramidal cell, spinal motoneuron, and retinal ganglion neurons) we found that SE of excitatory inputs decreases with the distance from the soma and active nonlinear properties of the dendrites can counterbalance this global effect of attenuation. This phenomenon is frequency dependent, with a more prominent gain in SE observed at lower levels of background input–output neuronal activity. In contrast, there are no significant differences in SE between passive and active dendrites under higher frequencies of background activity. The influence of the nonuniform distribution of active properties on SE is also more prominent at lower background frequencies. In models with real morphologies, the effect of active dendritic conductances becomes more dramatic and inverts the SE relationship between distal and proximal locations. In active dendrites, distal synapses have higher efficacy than that of proximal ones because of arising dendritic spiking in thin branches with high-input resistance. Lower levels of dendritic excitability can make SE independent of the distance from the soma. Although increasing dendritic excitability may boost SE of distal synapses in real neurons, it may actually reduce overall SE. The results are robust with respect to morphological variation and biophysical properties of the model neurons. The model of CA1 pyramidal cell with realistic distributions of dendritic conductances demonstrated important roles of hyperpolarization-activated (h-) current and A-type K+ current in controlling the efficacy of single synaptic inputs and overall SE differently in basal and apical dendrites.

An important consequence of gliotransmission, a signaling mechanism that involves glial release of active transmitter molecules, is its manifestation as N-methyl-d-aspartate receptor (NMDAR)-dependent slow inward currents in neurons. However, the intraneuronal spatial dynamics of these events or the role of active dendrites in regulating their amplitude and spatial spread have remained unexplored. Here, we used somatic and/or dendritic recordings from rat hippocampal pyramidal neurons and demonstrate that a majority of NMDAR-dependent spontaneous slow excitatory potentials (SEP) originate at dendritic locations and are significantly attenuated through their propagation across the neuronal arbor. We substantiated the astrocytic origin of SEPs through paired neuron–astrocyte recordings, where we found that specific infusion of inositol trisphosphate (InsP3) into either distal or proximal astrocytes enhanced the amplitude and frequency of neuronal SEPs. Importantly, SEPs recorded after InsP3 infusion into distal astrocytes exhibited significantly slower kinetics compared with those recorded after proximal infusion. Furthermore, using neuron-specific infusion of pharmacological agents and morphologically realistic conductance-based computational models, we demonstrate that dendritically expressed hyperpolarization-activated cyclic-nucleotide–gated (HCN) and transient potassium channels play critical roles in regulating the strength, kinetics, and compartmentalization of neuronal SEPs. Finally, through the application of subtype-specific receptor blockers during paired neuron–astrocyte recordings, we provide evidence that GluN2B- and GluN2D-containing NMDARs predominantly mediate perisomatic and dendritic SEPs, respectively. Our results unveil an important role for active dendrites in regulating the impact of gliotransmission on neurons and suggest astrocytes as a source of dendritic plateau potentials that have been implicated in localized plasticity and place cell formation.

Hippocampus, one of the parts included in the limbic system, is involved in various functions such as learning, memory, food-storing behavior, and sexual discrimination. Neuronal classes of the hippocampal complex in food-storing birds have been also reported, but the study lacks details pertaining to neuronal characteristics and the spine density of the neurons in different subfields of the hippocampus. Hence, the present study was undertaken with the aim to explore the morphology of neurons and the spines present on their dendrites within the hippocampal complex of the House Crow (Corvus splendens Vieillot, 1817), a food-storing Indian bird, and to compare it with previously reported nonfood-storing bird species. It was observed that the hippocampus of C. splendens harbors diverse neuronal classes with substantial percentages of pyramidal neurons, well-developed local circuit neurons, and high spine density. All these neuronal specializations in C. splendens can be related with the food-storing behavior of the bird, which itself is an advantage over nonfood-storing birds.

Abstract:In normal human aging the remaining neurons of two areas of the hippocampal region have been found to compensate for age-related neuronal loss by proliferating new dendrites. In Alzheimer's disease (AD) the layer II pyramidal neurons of the parahippocampal gyrus fail to show this compensatory response, in spite of a probable, exaggerated disease-related loss of neurons. In AD the dentate gyrus granule cells of the hippocampus also show a reduced amount of the compensatory response. This failure of the AD brain to show the normal compensatory plastic response, seen in normal aging as dendritic growth, may be viewed as one of the pathophysiological processes of the disease.

The sequential reactivation of memory-relevant neuronal ensembles during hippocampal sharp-wave (SW) ripple oscillations reflects cognitive processing. However, how a downstream neuron decodes this spatiotemporally organized activity remains unexplored. Using subcellular calcium imaging from CA1 pyramidal neurons in ex vivo hippocampal networks, we discovered that neighboring spines are activated serially along dendrites toward or away from cell bodies. Sequential spine activity was engaged repeatedly in different SWs in a complex manner. In a single SW event, multiple sequences appeared discretely in dendritic trees, but overall, sequences occurred preferentially in some dendritic branches. Thus, sequential replays of multineuronal spikes are distributed across several compartmentalized dendritic foci of a postsynaptic neuron, with their spatiotemporal features preserved.

Background: Neuronal plasticity is thought to underlie learning and memory formation. The density of dendritic spines in the CA1 region of the hippocampus has been repeatedly linked to mnemonic processes. Both the number and spatial location of the spines, in terms of proximity to nearest neighbour, have been implicated in memory formation. To examine how spatial training impacts synaptic structure in the hippocampus, Lister-Hooded rats were trained on a hippocampal-dependent spatial task in the radial-arm maze. Methods: One group of rats were trained on a hippocampal-dependent spatial task in the radial arm maze. Two further control groups were included: a yoked group which received the same sensorimotor stimulation in the radial-maze but without a memory load, and home-cage controls. At the end of behavioural training, the brains underwent Golgi staining. Spines on CA1 pyramidal neuron dendrites were imaged and quantitatively assessed to provide measures of density and distance from nearest neighbour. Results: There was no difference across behavioural groups either in terms of spine density or in the clustering of dendritic spines. Conclusions: Spatial learning is not always accompanied by changes in either the density or clustering of dendritic spines on the basal arbour of CA1 pyramidal neurons when assessed using Golgi imaging.

Background: Neuronal plasticity is thought to underlie learning and memory formation. The density of dendritic spines in the CA1 region of the hippocampus has been repeatedly linked to mnemonic processes. Both the number and spatial location of the spines, in terms of proximity to nearest neighbour, have been implicated in memory formation. To examine how spatial training impacts synaptic structure in the hippocampus, Lister-Hooded rats were trained on a hippocampal-dependent spatial task in the radial-arm maze. Methods: One group of rats were trained on a hippocampal-dependent spatial task in the radial arm maze. Two further control groups were included: a yoked group which received the same sensorimotor stimulation in the radial-maze but without a memory load, and home-cage controls. At the end of behavioural training, the brains underwent Golgi staining. Spines on CA1 pyramidal neuron dendrites were imaged and quantitatively assessed to provide measures of density and distance from nearest neighbour. Results: There was no difference across behavioural groups either in terms of spine density or in the clustering of dendritic spines. Conclusions: Spatial learning is not always accompanied by changes in either the density or clustering of dendritic spines on the basal arbour of CA1 pyramidal neurons when assessed using Golgi imaging.

The nervous system, including the brain, is a complex network with billions of complex neurons. Ion channels mediate the electrical signals that neurons use to integrate input and produce appropriate output, and could thus be thought of as key instruments in the neuronal orchestra. In the field of neuroscience we are not only curious about how our brains work, but also strive to characterize and develop treatments for neural disorders, in which the neuronal harmony is distorted. By modulating ion channel activity (pharmacologically or otherwise) it might be possible to effectively restore neuronal harmony in patients with various types of neural (including channelopathic) disorders. However, this exciting strategy is impeded by the gaps in our understanding of ion channels and neurons, so more research is required. Thus, the aim of this thesis is to improve the understanding of how specific ion channel types contribute to shaping neuronal dynamics, and in particular, neuronal integration, excitability and memory. For this purpose I have used computational modeling, an approach which has recently emerged as an excellent tool for understanding dynamically complex neurophysiological phenomena. In the first of two projects leading to this thesis, I studied how neurons in the brain, and in particular their dendritic structures, are able to integrate synaptic inputs arriving at low frequencies, in a behaviorally relevant range of ~8 Hz. Based on recent experimental data on synaptic transient receptor potential channels (TRPC), metabotropic glutamate receptor (mGluR) dynamics and glutamate decay times, I developed a novel model of the ion channel current ITRPC, the importance of which is clear but largely neglected due to an insufficient understanding of its activation mechanisms. We found that ITRPC, which is activated both synaptically (via mGluR) and intrinsically (via Ca2+) and has a long decay time constant (τdecay), is better suited than the classical rapidly decaying currents (IAMPA and INMDA) in supporting low-frequency temporal summation. It was further concluded that τdecay varies with stimulus duration and frequency, is linearly dependent on the maximal glutamate concentration, and might require a pair-pulse protocol to be properly assessed. In a follow-up study I investigated small-amplitude (a few mV) long-lasting (a few seconds) depolarizations in pyramidal neurons of the hippocampal cortex, a brain region important for memory and spatial navigation. In addition to confirming a previous hypothesis that these depolarizations involve an interplay of ITRPC and voltage-gated calcium channels, I showed that they are generated in distal dendrites, are intrinsically stable to weak excitatory and inhibitory synaptic input, and require spatial and temporal summation to occur. I further concluded that the existence of multiple stable states cannot be ruled out, and that, in spite of their small somatic amplitudes, these depolarizations may strongly modulate the probability of action potential generation. In the second project I studied the axonal mechanisms of unmyelinated peripheral (cutaneous) pain-sensing neurons (referred to as C-fiber nociceptors), which are involved in chronic pain. To my knowledge, the C-fiber model we developed for this purpose is unique in at least three ways, since it is multicompartmental, tuned from human microneurography (in vivo) data, and since it includes several biologically realistic ion channels, Na+/K+ concentration dynamics, a Na-K-pump, morphology and temperature dependence. Based on simulations aimed at elucidating the mechanisms underlying two clinically relevant phenomena, activity-dependent slowing (ADS) and recovery cycles (RC), we found an unexpected support for the involvement of intracellular Na+ in ADS and extracellular K+ in RC. We also found that the two major Na+ channels (NaV1.7 and NaV1.8) have opposite effects on RC. Furthermore, I showed that the differences between mechano-sensitive and mechano-insensitive C-fiber types might reside in differing ion channel densities. To conclude, the work of this thesis provides key insights into neuronal mechanisms with relevance for memory, pain and neural disorders, and at the same time demonstrates the advantage of using computational modeling as a tool for understanding and discovering fundamental properties of central and peripheral neurons.

QC 20120914

The electrophysiological properties of somatic and dendritic membranes of CA1 pyramidal neurons were investigated using the rat in vitro hippocampal slice preparation. A comprehensive analysis of extracellular field potentials, current-source density (CSD) and intracellular activity has served to identify the site of origin of action potential (AP) discharge in CA1 pyramidal neurons. 1) Action potential discharge of CA1 pyramidal cells was evoked by suprathreshold stimulation of the alveus (antidromic) or afferent synaptic inputs in stratum oriens (SO) or stratum radiatum (SR). Laminar profiles of the "stimulus evoked" extracellular field potentials were recorded at 25µm intervals along the dendro-somatic axis of the pyramidal cell and a 1-dimensional CSD analysis applied. 2) The shortest latency population spike response and current sink was recorded in stratum pyramidale or the proximal stratum oriens, a region corresponding to somata and axon hillocks of CA1 pyramidal neurons. A biphasic positive/negative spike potential (current source/sink) was recorded in dendritic regions, with both components increasing in peak latency through the dendritic field with distance from the border of stratum pyramidale. 3) A comparative intracellular analysis of evoked activity in somatic and dendritic membranes revealed a basic similarity in the pattern of AP discharge at all levels of the dendro-somatic axis. Stimulation of the alveus, SO, or SR evoked a single spike while injection of depolarizing current evoked a repetitive train of spikes grouped for comparative purposes into three basic patterns of AP discharge. 4) Both current and stimulus evoked intracellular spikes displayed a progressive decline in amplitude and increase in halfwidth with distance from the border of stratum pyramidale. 5) The only consistent voltage threshold for intracellular spike discharge was found in the region of the cell body, with no apparent threshold for spike activation in dendritic locations. 6) Stimulus evoked intradendritic spikes were evoked beyond the peak of the population spike recorded in stratum pyramidale, and aligned with the biphasic extradendritic field potential shown through laminar profile analysis to conduct with increasing latency from the cell body layer. The evoked characteristics of action potential discharge in CA1 pyramidal cells are interpreted to indicate the initial generation of a spike in the region of the soma-axon hillock and a subsequent retrograde spike invasion of dendritic arborizations.
Medicine, Faculty of
Cellular and Physiological Sciences, Department of
Graduate

Formins, also known as formin homology (FH) proteins, are involved in a wide range of actin-mediated processes. The Diaphanous-related formin Daam1 (Dishevelled-associated activator of morphogenesis) interacts with the PDZ domain protein Dishevelled, and is required to establish planar cell polarity in Xenopus. Through a yeast two-hybrid screen, I characterized a PDZ-mediated interaction between the C-terminus of Daam1 and the PDZ domains 456 of GRIP1. In dissociated rat hippocampal cultures, Daam1 expression was seen throughout the soma and dendrites in a punctate pattern. Furthermore, co-staining with a synaptic marker suggests that Daam1 could be associated with post-synaptic specializations. Dendritic spines are enriched with actin filaments, and based on the subcellular localization of Daam1 and the evidence that formins are involved in regulating actin polymerization, I hypothesized that Daam1 might play a role in dendritic spine morphology. In order to investigate the functional roles for Daam1, viral vectors were developed using the Semliki-Forest defective viral vector to over-express the full-length Daam1 protein and a Daam1 lacking the PDZ-binding motif. The over-expression of the full-length Daam1 in organotypic hippocampal slices showed a punctate distribution throughout the dendritic shaft, with the occasional appearance in spines, resulting in an overall increase in dendritic spine length. This suggests that formins, such as Daam1, could potentially regulate spine morphology.

Kainic acid induced excitotoxicity causes pyramidal cell death in the CA3a/b region of the hippocampus. Electrical synapses, gap junctional communication, and single membrane channels in non-junctional membranes (hemichannels) composed of connexin36 (Cx36) have been implicated in both seizure propagation and the spread of excitotoxic cell death. In rats, Cx36 protein is expressed by pyramidal neurons. Localization of protein in mouse, however, is highly controversial. Expression is reported to be restricted to hippocampal interneurons yet the same excitotoxic mechanisms (electrical and metabolic coupling between pyramidal neurons) are invoked to explain the role of Cx36 in excitotoxic pyramidal loss in murine brain. To address this controversy, I show by confocal immunofluorescence and in situ hybridization that Cx36 protein expression is restricted to interneurons and microglia in murine hippocampus and is not expressed by, or is below level of detection in pyramidal neurons. Using behavioural and electrophysiological measures, seizure propagation was found to be moderately enhanced in the absence of Cx36 likely due to the loss of interneuron-mediated synchronous inhibition of the pyramidal cells. Further, CA3a/b neurons die post kainic acid injury in the presence of Cx36 but are protected in Cx36-/- mice. When delayed excitotoxic cell death is maximal, Cx36 is primarily expressed by activated microglia as demonstrated by confocal immunofluorescence, in situ hybridization, and Western blotting. These activated microglia are located in the direct vicinity of, and surrounding cells in the damaged Ca3a/b region. Finally, I show that loss of Cx36 from activated microglia in mice is sufficient to prevent excitotoxic cell death in the CA3a/b with surviving neurons functional as assessed by both electrophysiological and behavioural measures. Together, these data identify a new mechanism of excitotoxic injury, mediated by neuronal-glial interactions, and dependent on microglial Cx36 expression.

Thesis (Ph.D.)--University of Toledo, 2007.
"In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences." Major advisor: Elizabeth Tietz. Includes abstract. Title from title page of PDF document. Bibliography: pages 88-94, 130-136, 178-189, 218-266.

Das deklarative Gedächtnis beruht auf Wechselwirkungen zwischen dem medialen Temporallappens (MTL) und Neokortex. Aufgrund der verteilten Natur neokortikaler Netzwerke bleiben zelluläre Ziele und Mechanismen der Gedächtnisbildung im Neokortex jedoch schwer fassbar. Im sechsschichtigen Säugetier-Neokortex konvergieren die Top-Down-Inputs auf Schicht 1 (L1). Wir untersuchten, wie Top-Down-Inputs von MTL die neokortikale Aktivität während der Gedächtnisbildung modulieren. Wir haben zunächst ein Kortex- und Hippocampus-abhängiges Lernparadigma angepasst, in dem Tiere gelernt haben, direkte kortikale Mikrostimulation und Belohnung zu assoziieren. Neuronen in den tiefen Schichten des perirhinalen Kortex lieferten monosynaptische Eingaben in L1 des primären somatosensorischen Kortex (S1), wo die Mikrostimulation vorgestellt wurde. Die chemogenetische Unterdrückung der perirhinalen Inputs in L1 von S1 störte die Gedächtnisbildung, hatte jedoch keinen Einfluss auf die Leistung der Tiere nach abgeschlossenem Lernen. Dem Lernen folgte das Auftreten einer klaren Subpopulation von Pyramidenneuronen der Schicht 5 (L5), die durch hochfrequentes Burst-Feuern gekennzeichnet war und durch Blockieren der perirhinalen Inputs zu L1 reduziert werden konnte. Interessanterweise zeigte ein ähnlicher Anteil an apikalen Dendriten von L5-Pyramidenneuronen ebenfalls eine signifikant erhöhte Ca2+-Aktivität während des Gedächtnisabrufs bei Expertentieren. Wichtig ist, dass die Störung der dendritischen Ca2+-Aktivität das Lernen beeinträchtigte, was darauf hindeutet, dass apikale Dendriten von L5-Pyramidenneuronen eine entscheidende Rolle bei der Bildung des neokortikalen Gedächtnisses spielen. Wir schließen daraus, dass MTL-Eingaben das Lernen über einen perirhinalen vermittelten Gating-Prozess in L1 steuern, der sich in einer erhöhten dendritischen Ca2+-Aktivität und einem Burst-Firing in pyramidalen L5-Neuronen manifestiert.
Declarative memory relies on interactions between the medial temporal lobe (MTL) and neocortex. However, due the distributed nature of neocortical networks, cellular targets and mechanisms of memory formation in the neocortex remain elusive. In the six-layered mammalian neocortex, top-down inputs converge on its outermost layer, layer 1 (L1). We examined how layer-specific top-down inputs from MTL modulate neocortical activity during memory formation. We first adapted a cortical- and hippocampal-dependent learning paradigm, in which animals learned to associate direct cortical microstimulation and reward, and characterized the learning behavior of rats and mice. We next showed that neurons in the deep layers of the perirhinal cortex not only provide monosynaptic inputs to L1 of the primary somatosensory cortex (S1), where microstimulation was presented, but also actively reflect the behavioral outcome. Chemogenetic suppression of perirhinal inputs to L1 of S1 disrupted early memory formation but did not affect animals’ performance after learning. The learning was followed by an emergence of a distinct subpopulation of layer 5 (L5) pyramidal neurons characterized by high-frequency burst firing, which could be reduced by blocking perirhinal inputs to L1. Interestingly, a similar proportion of apical dendrites (~10%) of L5 pyramidal neurons also displayed significantly enhanced calcium (Ca2+) activity during memory retrieval in expert animals. Importantly, disrupting dendritic Ca2+ activity impaired learning, suggesting that apical dendrites of L5 pyramidal neurons have a critical role in neocortical memory formation. Taken together, these results suggest that MTL inputs control learning via a perirhinal-mediated gating process in L1, manifested by elevated dendritic Ca2+ activity and burst firing in L5 pyramidal neurons. The present study provides insights into cellular mechanisms of learning and memory representations in the neocortex.

In the pursuit to understand information processing in the brain, a general question that is often posed is: What is the neuron encoding? This is addressed by assessing feature selectivity in sensory neurons, which informs us of the specific features of the stimulus that the neuron is selectively responding to. In this respect, a quantity that has been widely used is the spike triggered average (STA), an unbiased estimate of the spike-triggering features in the stimulus. From the single neuron perspective, the STA provides us with a direct link connecting the biophysical properties of the neuron and its response dynamics in a network with its encoding schema. While this approach has been used extensively for sensory modalities, it is a slightly more complex problem for multimodal areas such as the hippocampus where the information itself is in the form of complex spatiotemporal pattern of synaptic inputs. The hippocampus is a brain region that has been implicated in navigation, learning and encoding of several forms of context-dependent and episodic memory. Pyramidal neurons in the CA1 subfield of the hippocampus receive synaptic inputs from the entorhinal cortex and the CA3 pyramidal neurons. These inputs are spatially segregated on the dendritic arbour of CA1 pyramidal neurons as well as containing information in spectrally parsed streams. Specifically, inputs from the entorhinal cortex exhibit high power in the fast gamma frequency range (60–100 Hz), whereas inputs from the CA3 exhibit high power in the slow gamma frequency range (30–60 Hz). The spectral segregation of inputs has important ramifications for encoding of specific, behaviourally relevant information about the space and contextual cues in an ambulatory animal. Moreover, these neurons exhibit a form of multiplexed coding where the firing rate while expressing coherence with different gamma frequency bands also demonstrate phase-locking with an ongoing slower oscillation in the theta (4–10 Hz) frequency range. Research spanning the past two decades has resulted in extensive literature on the active and passive properties of the CA1 pyramidal neuronal dendrites, their somato-dendritic transfer properties and the physiological roles of the heterogeneous distribution of voltage-gated ion channels along their somato-dendritic arbour. In addition, location-dependence of several physiological properties including subthreshold theta resonance and phase preference in these neurons, as well as activity-dependent plasticity in ion channel expression profiles and subsequent plasticity in their excitability and impedance characteristics have also been established in these neurons. Juxtaposed against this extensive literature on active dendritic physiology and plasticity with specific reference to hippocampal pyramidal neurons, we asked the following questions. 1. What feature selectivity does the spike initiation dynamics of CA1 pyramidal neurons exhibit? Specifically, these neurons are known to exhibit theta-frequency subthreshold resonance. But does this subthreshold resonance also translate to spectral selectivity in the suprathreshold regime manifesting as theta-frequency selectivity in their spike initiation dynamics? 2. Given the intraneuronal gradients in various ion channel properties and in physiological properties regulated by these ion channels, is there also a location-dependent gradient, or an intraneuronal functional map, in the spike initiation dynamics of these neurons? 3. Do CA1 pyramidal neurons exhibit coincidence detection of excitatory synaptic inputs in the gamma frequency range? Given the spatial segregation of the spectrally parsed afferent inputs along the CA1 apical dendrites, is the coincidence detection window expressed by these neurons equipped to detect these differences in afferent frequencies in a location-dependent manner? 4. How do plastic active dendrites regulate location dependent spike initiation dynamics and coincidence detection windows? How do active properties of the dendrites and plasticity therein alter the location dependence of spike initiation dynamics? 5. How do the somato-dendritic ion channels regulate the specific spike-triggering features in CA1 pyramidal neurons? How do individual channels and interactions among them alter the location dependence of spike initiation dynamics and coincidence detection windows? 6. Are there correlations between the frequency selectivity in spike initiation dynamics and subthreshold resonance frequency? Under what conditions do these correlations hold and when does the correlation disintegrate? Are there specific ion channels that mediate correlations between the frequency selectivity in the sub- and supra-threshold regimes? In the pursuit to answer these questions, we employed a combination of computational and electrophysiological methods to assess the spike initiation dynamics and coincidence detection windows in CA1 pyramidal neurons using the STA and various STA-derived quantitative metrics that we developed as part of this thesis. As a first step, we employed a single compartmental model of a CA1 pyramidal neuron with only the spiking conductances (transient sodium and delayed rectifier potassium channels) and the hyperpolarization-activated cyclic nucleotide-gated (HCN) nonspecific cation channel, an established mediator of subthreshold resonance (fR) in these neurons. We injected zero-mean Gaussian white noise (GWN) current with a fixed standard deviation adjusted to elicit ~1–2 Hz average firing. We measured the STA as the average current in a 1 s time window preceding a spike computed over ~1000 spikes obtained in response to the GWN current. We computed the STA for various densities of HCN channels and observed that the STA transitions from class I in the absence of HCN channels to class II in their presence. This was evidenced by an increase in the depth of the negative lobe as well as a progressive sharpening/narrowing of the spike-proximal positive lobe (SPPL) with increasing density of HCN channels. To systematically quantify these changes, we developed specific metrics based on the shape of the STA. First, the peak of the positive lobe of the STA was quantified and was found to increase with increasing HCN conductance suggesting an inverse relationship between this peak and the excitability of the neuron. Next, we performed Fourier transform on the STA, which revealed spectral selectivity in the STA defined by a distinct band-pass structure in the frequency domain. We quantified the frequency at which the |STA(f)| reached its maximum as the STA characteristic frequency (fSTA) and found this to be in the theta frequency range in the presence of HCN channels, with increase in the fSTA on increasing HCN conductance. Furthermore, the strength of this frequency selectivity (QSTA), quantified as the ratio |STA(f)|/ |STA(0.5 Hz)| also increased with HCN density. We reasoned that the SPPL of the STA reflects the temporal window over which the neuron integrates coincident inputs and so quantified the total duration of the SPPL as the total coincidence detection window (TTCDW). Additionally, to account for the shape of the STA that underwent a change on altering HCN conductance, we computed the effective coincidence detection window (TECDW) as the STA-weighted measure of the SPPL. Both the total and effective CDW underwent a reduction on increasing the HCN channel density and the effective coincidence detection window was in the gamma frequency range. Together, these results demonstrated that the HCN channel alone was sufficient to confer coincidence detector properties on the neuron in the gamma frequency range as well as theta frequency selectivity in the spike initiation dynamics. This also implied that graded expression of HCN channels was sufficient to effectuate a transition in the STA along the integrator-coincidence detector (I-CD) continuum. We confirmed that such a transition could not be elicited simply by altering the passive properties of the membrane, in particular the leak conductance, and was specifically mediated by HCN channels. Further analysis in the single compartmental model suggested that the fSTA was correlated to the fR in the presence of HCN channels. However, in their absence, there was delta frequency selectivity in the STA, which was critically dependent upon the density and kinetics of the spiking conductances. Importantly, although subthreshold resonance was completely abolished in the absence of HCN channels, the transient sodium channels and delayed rectifier potassium channels mediated delta frequency selectivity in the STA, thereby providing lines of evidence on a dissociation between subthreshold resonance and STA spectral selectivity. Next, to assess location dependence of the STA, we built models of increasing complexity from a ball-and-stick model to a morphologically realistic model and measured the STA (with reference to somatic spike timings) by injecting GWN at various locations along the dendrite. We imposed a gradient of HCN channels on these models that matched electrophysiological data on gradients in input resistance and resonance frequency and assessed the distance-dependent variation in STA measurements. We found that in the ball-and-stick model with a non-spiking dendrite, the fSTA was normalized with distance from soma and global plasticity in HCN channel density altered fSTA values across locations in a distance-invariant manner. Introduction of spike-generating conductances into the dendritic compartment resulted in enhancements of fSTA and QSTA, with distance-invariance perturbed at distal dendritic locations owing to dendritic spike initiation. A morphologically realistic model, on the other hand, exhibited a clear functional map in the STA with both fSTA and QSTA increasing with distance, suggesting that the STA was dependent on the location of inputs along the somato-dendritic axis. Thus far, our focus was on the HCN channel and its interactions with the spike generating conductances in regulating STA measurements. In assessing the impact of other channels that are expressed by CA1 pyramidal neurons, we first employed single compartmental models to study the effect of kinetic interactions between these ion channels. These analyses revealed that the co-presence of another resonating conductance, the T-type calcium (CaT) channel, further increased the fSTA and QSTA in the presence of HCN channels while also reducing the CDW measures. However, the correlation between the fSTA and fR was reduced in the presence of CaT channels. On the other hand, the co-presence of HCN channels with a subthreshold restorative conductance, mediated by A-type potassium channels, reduced the fSTA and QSTA but broadened the CDW. A regenerative conductance, the persistent sodium channel increased the QSTA significantly without significantly altering fSTA. These observations clearly dissected the differential effects of various ion channels and interactions therein on STA and CDWs. They also demonstrated that a clear correlation between sub- and supra-threshold frequency selectivity existed only when HCN channels were the sole subthreshold channels present in the model. However, when multiple ion channels came into play or when HCN channels were absent, a clear dissociation between these forms of selectivity was observed. To assess the role of active plastic dendrites spanning several somatodendritic ion channels, we incorporated gradients in HCN, CaT and KA into a morphologically realistic model and tuned several measurements to match electrophysiological data. In this model, we observed the emergence of location dependent theta frequency selectivity in STA showing strong class II characteristics. Importantly, a gradient in CDW measures was also observed, with the effective CDW decreasing from slow gamma range in the proximal dendrites to fast gamma range in the distal dendrites. Removal of all active conductances resulted in a transition to class I STA with significant broadening of the integration window, and the presence of uniform resonating conductances in the dendrites resulted in class I STA with a narrow integration window. These results demonstrated the emergence of location-dependent theta frequency selectivity in the STA and the presence of stratified gamma-range CDW that is essential for detecting frequency-multiplexed inputs afferent onto different regions of the dendritic arbor. These observations also emphasized the importance of gradients in ion channels in maintaining functional maps of spike initiation dynamics and CDW, and suggested that local or global plasticity in any or all of these ion channels would alter feature selectivity and coincidence detection in hippocampal pyramidal neurons. We finally tested our computational predictions using acute rodent hippocampal slices and performed electrophysiological measurements of somatic STA from CA1 pyramidal neurons. Our primary goals were to (i) confirm theta frequency selectivity and gamma-range CDW in the STA of CA1 pyramidal neurons as predicted from our models; (ii) test the quantitative prediction that the blockade of HCN channels would reduce fSTA from theta- to delta-frequency ranges; and (iii) assess the efficacy of the novel STA-derived metrics against other physiological measurements of excitability and impedance profiles from the same neurons. Our results unveiled theta-frequency selectivity in the STA of hippocampal pyramidal neuron somata, coupled with gamma-range CDW, thereby validating our computational predictions. Furthermore, we confirmed that the correlation between the fSTA and fR was weak across cells, corroborating our model prediction on the dissociation between the two forms of spectral selectivity in the presence of multiple ion channels. We observed strong correlations between the peak STA current and excitability measures such as input resistance and impedance amplitude, as well as strong correlations between the CDW measures and fR suggesting that neurons resonant at higher frequencies could detect coincident inputs at higher frequencies. We also demonstrated the dependence of the STA measurements on the membrane potential with hyperpolarization increasing the fSTA akin to increase in HCN-dependent fR, but the QSTA increased on depolarization. Our results also revealed the adaptability of the somatic STA and its quantitative measurements to the input statistics. Finally, we pharmacologically tested the impact of blocking HCN channels on somatic STA of identified hippocampal pyramidal neurons. We found, consistent with our model predictions, that fSTA decreased from theta frequency to delta frequency upon blockade of HCN channels, although subthreshold resonance was completely abolished with HCN-channel blockade. Our electrophysiological analyses allowed us to confirm many of our model predictions, apart from demonstrating HCN-channel dependent theta-frequency selectivity in spike initiation dynamics and gamma-range coincidence detection windows in CA1 pyramidal neurons. Together, the results of our computational and electrophysiological studies unveiled the critical role of several voltage-gated ion channels in regulating spectral selectivity in spike initiation dynamics as well as in mediating sharp coincidence detection windows. Our computational analyses expounded the role of plastic active dendrites in mediating a functional map in the STA in CA1 pyramidal neurons, with multiple, degenerate mechanisms involving interactions between ion-channels and a state-dependent modulation of the STA. These results identified explicit roles for plastic active dendrites in neural coding and strongly recommend a dynamically reconfigurable multi-STA model to characterize location-dependent input feature selectivity in pyramidal neurons. Importantly, they also showed that the presence of resonating and spike-generating conductances serve as a mechanism underlying the emergence of stratified gamma-range coincidence detection in the dendrites of CA1 pyramidal neurons, enabling them to perform behaviour- and state-dependent gamma-frequency multiplexing. Our electrophysiological experiments confirmed many of our model predictions including the critical role of HCN channels in mediating theta-frequency selectivity and paved the way for future studies involving dendritic ion channels and plasticity therein. The thesis is organized into a total of eight chapters with Chapter 1 providing a general introduction and laying the motivations for the thesis. Chapter 2 is an overview of literature on the physiology of the hippocampus that served as the cornerstone for the pursuits of this thesis. Chapter 3 is a brief discourse on the theoretical principles and computational models of single neurons that were paramount to synthesizing this thesis. Chapter 4 is the first of the results chapters and discusses the various STA-derived metrics that were used to quantify spectral selectivity and coincidence detection windows in the STA of model hippocampal pyramidal neurons along with dissecting the specific role of resonating conductances in mediating these forms of feature selectivity. Chapter 5 explores the role of dendrites in mediating location dependence in the STA, focusing specifically on theta frequency selectivity in STA and interactions between HCN channels and the spiking conductances. The chapter also establishes specific correlations and dissociations between sub- and supra-threshold theta frequency selectivity. Chapter 6 first delves into the kinetic interactions between various dendritically expressed subthreshold conductances in a single-compartmental model. The latter part of this chapter assesses spatio-temporal interactions between these conductances in a morphologically precise model towards concerted regulation of location-dependent STA and coincidence detection windows. Chapter 7 presents the results of electrophysiological experiments on rodent acute hippocampal slices, demonstrating theta frequency selectivity and gamma-range coincidence detection window in the somatic STA of CA1 pyramidal neurons. The chapter also confirms computational predictions on the reduction of STA frequency selectivity from theta to delta ranges upon pharmacological blockade of HCN channels. Finally, Chapter 8 presents the broad implications of results presented here and posit some future directions stemming from this thesis.

Intricate interactions among constituent components are defining hallmarks of biological systems and sculpt physiology across different scales spanning gene networks to behavioural repertoires. Whereas interactions among channels and receptors define neuronal physiology, interactions among different cells specify the characteristic features of network physiology. From a single-neuron perspective, it is now evident that the somato-dendritic plasma membrane of hippocampus pyramidal neurons is endowed with several voltage-gated ion channels (VGICs) with varying biophysical properties and sub cellular expression profiles. Structural and physiological interactions among these channels define generation and propagation of electrical signals, thereby transforming neuronal dendrites to actively excitable membrane endowed with complex computational capabilities. In parallel to this complex network of plasma membrane channels is an elegantly placed continuous intraneuronal membrane of the endoplasmic reticulum (ER) that runs throughout the neuronal morphology. Akin to the plasma membrane, the ER is also endowed with a variety of channels and receptors, prominent among them being the inositol trisphosphate (InsP3) receptors (InsP3Rs) and ryanodine receptors (RyR), both of which are calcium release channels. Physiological interactions among these receptors transform the ER into a calcium excitable membrane, capable of active propagation of calcium waves and of spatiotemporal integration of neuronal signals. Thus, a neuron is endowed with two continuously parallel excitable membranes that actively participate in the bidirectional flow of intraneuronal information, through interactions among different channels and receptors on either membrane. Although the interactions among sets of channels and receptors present individually on either membrane are very well characterized, our understanding of cross-membrane interactions among channels and receptors across these two membranes has been very limited. Recent literature has emphasized the critical nature of such cross-membrane interactions and the several physiological roles played by such interactions. Such cross-channel interactions include ER depletion-induced signaling involving store-operated calcium channels, generation and propagation of calcium waves through interactions between plasma membrane and ER membrane receptors, and the plasticity of plasma membrane VGICs and receptors induced by ER Ca2+. Such tight interactions between these two membranes have highlighted several roles of the ER in the integration of intraneuronal information, in regulating signalling microdomains and in regulating the downstream signaling pathways that are regulated by these Ca2+ signals. Yet, our understanding about the functional interactions between the ion channels and receptors present on either of these membranes is very limited from the perspective of the combinatorial possibilities that encompass the span of channels and receptors across these two membranes. In this context, the first part of this thesis deals with two specific instances of such cross-membrane functional interactions, presented as two subparts with each probing different direction of impact. Specifically, whereas the first of these subparts deals with the impact of plasma membrane VGICs on the physiology of ER receptors, the second subpart presents an instance of the effect of ER receptor activation on plasma membrane VGIC. In the first subpart of the thesis, we establish a novel role for the A-type potassium current in regulating the release of calcium through inositol triphosphate receptors (InsP3R) that reside on the endoplasmic reticulum (ER) of hippocampus pyramidal neurons. Specifically, the A-type potassium current has been implicated in the regulation of several physiological processes including the regulation of calcium influx through voltage-gated calcium channels (VGCCs). Given the dependence of InsP3R open probability on cytosolic calcium concentration ([Ca2+]c) we asked if this regulation of calcium influx by A-type potassium current could translate into the regulation of release of calcium through InsP3Rs by the A-type potassium current. To answer this, we constructed morphologically realistic, conductance-based neuronal models equipped with kinetic schemes that govern several calcium signalling modules and pathways, and constrained the distributions and properties of constitutive components by experimental measurements from these neurons. Employing these models, we establish a bell-shaped dependence of calcium release through InsP3Rs on the density of A-type potassium current, during the propagation of an intraneuronal calcium wave initiated through established protocols. Exploring the sensitivities of calcium wave initiation and propagation to several underlying parameters, we found that ER calcium release critically depends on dendrite diameter and wave initiation occurred at branch points as a consequence of high surface area to volume ratio of oblique dendrites. Further, analogous to the role of A-type potassium channels in regulating spike latency, we found that an increase in the density of A-type potassium channels led to increases in the latency and the temporal spread of a propagating calcium wave. Next, we incorporated kinetic models for the metabotropic glutamate receptor (miler) signalling components and a calcium-controlled plasticity rule into our model and demonstrate that the presence of mGluRs induced a leftward shift in a BCM-like synaptic plasticity profile. Finally, we show that the A-type potassium current could regulate the relative contribution of ER calcium to synaptic plasticity induced either through 900 pulses of various stimulus frequencies or through theta burst stimulation. These results establish a novel form of interaction between active dendrites and the ER membrane and suggest that A-type K+ channels are ideally placed for inhibiting the suppression of InsP3Rs in thin-caliber dendrites. Furthermore, they uncover a powerful mechanism that could regulate biophysical/biochemical signal integration and steer the spatiotemporal spread of signalling micro domains through changes in dendritic excitability. In the second subpart, we turned our focus to the role of calcium released through InsP3Rs in regulating the properties of VGICs present on the plasma membrane, thereby altering neuronal intrinsic properties that are dependent on these VGICs. Specifically, the synaptic plasticity literature has focused on establishing necessity and sufficiency as two essential and distinct features in causally relating a signalling molecule to plasticity induction, an approach that has been surprisingly lacking in the intrinsic plasticity literature. Here, we complemented the recently established necessity of inositol trisphosphate (InsP3) receptors (InsP3R) in a form of intrinsic plasticity by asking if ER InsP3R activation was sufficient to induce plasticity in intrinsic properties of hippocampus neurons. To do this, we employed whole-cell patch-clamp recordings to infuse D-myo-InsP3, the endogenous ligand for InsP3Rs, into hippocampus pyramidal neurons and assessed the impact of InsP3R activation on neuronal intrinsic properties. We found that such activation reduced input resistance, maximal impedance amplitude and temporal summation, but increased resonance frequency, resonance strength, sag ratio, and impedance phase lead of hippocampus pyramidal neurons. Strikingly, the magnitude of plasticity in all these measurements was dependent upon [InsP3], emphasizing the graded dependence of such plasticity on InsP3R activation. Mechanistically, we found that this InsP3-induced plasticity depended on hyperpolarization-activated cyclic-nucleotide gated (HCN) channels. Moreover, this calcium-dependent form of plasticity was critically reliant on the release of calcium through InsP3Rs, the influx of calcium through N-methyl-D -aspartate receptors and voltage-gated calcium channels, and on the protein kinase A pathway. These results delineate a causal role for InsP3Rs in graded adaptation of neuronal response dynamics through changes in plasma membrane ion channels, thereby revealing novel regulatory roles for the endoplasmic reticulum in neural coding and homeostasis. Whereas the first part of the thesis dealt with bidirectional interactions between ER membrane and plasma membrane channels/receptors within a neuron, second part focuses on cross-cellular interactions, specifically between ER membrane on astrocytes and dendritic plasma membrane of neurons. Specifically, the universality of ER-dependent calcium signalling ensures that its critical influence extends to regulating the physiology of astrocytes, an abundant form of glial cells in the hippocampus. Due to the presence of calcium release channels on ER membrane, astrocytes are calcium excitable, whereby they respond to neuronal activity by increase in their cytosolic calcium levels. Specifically, astrocytes respond to the release of neurotransmitters from neuronal presynaptic terminals through activation of metabotropic receptors expressed on their plasma membrane. Such activation results in the mobilization of cytosolic InsP3 and subsequent release of calcium through InsP3 on the astrocytes ER membrane. These ER-dependent [Ca2+]c elevations in astrocytes then result in the release of gliotransmitters from astrocytes, which bind to corresponding receptors located on neuronal plasma membrane resulting in voltage-deflections and/or activation of signaling pathways in the neuron. Although it is well established that gliotransmission constitutes an important communication channel between astrocytes and neurons, the impact of gliotransmission on neurons have largely been centered at the cell body of the neurons. Consequently, the impact of the activation of astrocytic InsP3R on neuronal dendrites, and the role of dendritic active conductances in regulating this impact have been lacking. This lacuna in mapping the spatial spread of gliotransmission in neurons is especially striking because most afferent synapses impinge on neuronal dendrites, and a significant proportion of information processing in neurons is performed in their dendritic arborization. Additionally, given that active dendritic conductances play a pivotal role in regulating the impact of fast synaptic neurotransmission on neurons, we hypothesized that such active-dendritic regulation should extend to the impact of slower extrasynaptic gliotransmission on neurons. The second part of the thesis is devoted to testing this hypothesis using dendritic and paired astrocyte-neuron electrophysiological recordings, where we also investigate the specific roles of active dendritic conductances in regulating the impact of gliotransmission initiated through activation of astrocytic InsP3Rs. In testing this hypothesis, in the second part of the thesis, we first demonstrate a significantly large increase in the amplitude of astrocytically originating spontaneous slow excitatory potentials (SEP) in distal dendrites compared to their perisomatic counterparts. Employing specific neuronal infusion of pharmacological agents, we show that blocking HCN channels increased the frequency, rise-time and width of dendritically-recorded spontaneous SEPs, whereas blockade of A-type potassium channels enhanced their amplitude. Next, through paired neuron-astrocytes recordings, we show that our conclusions on the differential roles of HCN and A-type potassium channels in modulating spontaneous SEPs also extended to SEPs induced through infusion of InsP3 in a nearby astrocyte. Additionally, employing subtype-specific receptor blockers during paired neuron-astrocyte recordings, we provide evidence that GluN2B-and GluN2D-containing NMDARs predominantly mediate perisomatic and dendritic SEPs, respectively. Finally, using morphologically realistic conductance-based computational models, we quantitatively demonstrate that dendritic conductances play an active role in mediating compartmentalization of the neuronal impact of gliotransmission. These results unveil an important role for active dendrites in regulating the impact of gliotransmission on neurons, and suggest astrocytes as a source of dendritic plateau potentials that have been implicated in localized plasticity and place cell formation. This thesis is organized into six chapters as follows: Chapter 1 lays the motivations for the questions addressed in the thesis apart from providing the highlights of the results presented here. Chapter 2 provides the background literature for the thesis, introducing facts and concepts that forms the foundation on which the rest of the chapters are built upon. In chapter 3, we present quantitative analyses of the physiological interactions between A-type potassium conductances and InsP3Rs in CA1 pyramidal neurons. In chapter 4, using electrophysiological recordings, we investigate the role of calcium released through InsP3Rs in induction of plasticity of intrinsic response dynamics, and demonstrate that this form of plasticity is consequent to changes in neuronal HCN channels. In chapter 5, we systematically map the spatial dynamics of the impact of gliotransmission on neurons across the somato-apical trunk, also unveiling the role of neuronal HCN and A-type potassium channels in compartmentalizing such impact. Finally, chapter 6 concludes the thesis highlighting its major contributions and discussing directions of future research.

Vanadium (V), a widely distributed transition metal, has been considered toxic, which depends on the valence of the compound. V pentoxide (V2O5) is considered the most harmful. Its long-term exposure produces neurotoxicity. Mice exposed to inhaled V2O5 displayed less tubulin+ in testicular cells and dendritic spines loss, cell death, and CA1 neuropil modifications, considered as the result of V interaction with the cytoskeleton, which made us suppose that V2O5 inhalation could initiate CA1 cell alterations comparable to what happen in the brains of Alzheimer disease (AD) patients. This study intends to demonstrate pyramidal CA1 cytoskeletal changes in rats which inhaled V2O5. Twenty rats were exposed to V2O5 0.02 M one hour, three times a week for several months. Our findings showed that V2O5-exposed rats had cell death that reached 56,57% after six months; we also observed collapsed strong argyrophilic nuclei and characteristic flame-shaped somas in all V2O5-exposed animals hippocampus CA1 compared to controls. We also found somatodendritic deformations. Neurite’s cytoskeleton exhibited visible thickening and nodosities and prominent dendritic spine loss. Our results demonstrate that V2O5 induces AD-like cell death with evident cytoskeletal and synaptic alterations.

Abstract The Orch OR theory attributes consciousness to “orchestrated” quantum computations in microtubules inside brain neurons. These terminate by Penrose objective reduction (OR), a process in the fine scale structure of the universe which introduces phenomenal experience and non-computability. Lattice polymers of tubulin proteins, microtubules organize intra-neuronal activities. The Penrose-Hameroff Orch OR theory suggests that microtubules 1) encode memory and process information, 2) orchestrate quantum vibrational superpositions (qubits) of pi electron resonance dipoles within tubulin which unify, entangle and 3) evolve to meet Orch OR threshold for full, rich conscious experience, most likely 4) in dendrites and soma of cortical layer 5 pyramidal neurons, and 5) selection of microtubule states which regulate axonal firings and behavior. Penrose has further described 6) retroactivity inherent in OR and Orch OR, which can resolve Libet’s backward time referral, and rescue conscious free will. Orch OR has explanatory power, and is testable and falsifiable.

Nerve cells are the targets of many thousands of excitatory and inhibitory synapses. An extreme case are the Purkinje cells in the primate cerebellum, which receive between one and two hundred thousand synapses onto dendritic spines from an equal number of parallel fibers (Braitenberg and Atwood, 1958; Llinas and Walton, 1998). In fact, this structure has a crystalline-like quality to it, with each parallel fiber making exactly one synapse onto a spine of a Purkinje cell. For neocortical pyramidal cells, the total number of afferent synapses is about an order of magnitude lower (Larkman, 1991). These numbers need to be compared against the connectivity in the central processing unit (CPU) of modern computers, where the gate of a typical transistor usually receives input from one, two, or three other transistors or connects to one, two, or three other transistor gates. The large number of synapses converging onto a single cell provide the nervous system with a rich substratum for implementing a very large class of linear and nonlinear neuronal operations. As we discussed in the introductory chapter, it is only these latter ones, such as multiplication or a threshold operation, which are responsible for “computing” in the nontrivial sense of information processing. It therefore becomes crucial to study the nature of the interaction among two or more synaptic inputs located in the dendritic tree. Here, we restrict ourselves to passive dendritic trees, that is, to dendrites that do not contain voltage-dependent membrane conductances. While such an assumption seemed reasonable 20 or even 10 years ago, we now know that the dendritic trees of many, if not most, cells contain significant nonlinearities, including the ability to generate fast or slow all-or-none electrical events, so-called dendritic spikes. Indeed, truly passive dendrites may be the exception rather than the rule in the nervous In Sec. 1.5, we studied this interaction for the membrane patch model. With the addition of the dendritic tree, the nervous system has many more degrees of freedom to make use of, and the strength of the interaction depends on the relative spatial positioning, as we will see now. That this can be put to good use by the nervous system is shown by the following experimental observation and simple model.

Laser scanning microscopy combined with two-photon excitation of fluorescence holds great promise in imaging biological systems. This two-photon excitation laser scanning microscopy (TPLSM) [1] yields intrinsic submicron three-dimensional resolution with much reduced background fluorescence and thus reduced photodamage. Although the advantages of TPLSM as compared to wide field fluorescence microscopy and confocal microscopy have been demonstrated in a number of applications [2], the large cost and utility requirements of mode locked Ti:sapphire laser systems and other femtosecond light sources have kept TPLSM out of reach for most biology labs. We demonstrate here that a recently developed compact solid state laser that is mode locked with a Saturable Bragg Reflector (SBR) [3] is well-suited for TPLSM. A SBR-modelocked Cr:LiSAF laser was pumped with a 0.5 W, 670 nm diffraction-limited MOPA (SDL), providing 90 fs pulses at 860 nm with CW power of 25-44 mW per beam (Fig. la). A single beam was directed into a laser scanning microscope consisting of a pair of galvomirrors, a relay lens, a dichroic mirror, a Zeiss water-immersion objective (63 x 0.9 NA), and a photomultiplier tube for the detection of fluorescence photons [2]. Rat cortical brain slices (300 μm thick) were prepared using standard techniques. For anatomical imaging, neocortical pyramidal cells that were deeply embedded in the tissue were dialyzed and voltage clamped using whole-cell electrodes containing 500 μM fluorescein dextran (MW = 3 kD). TPLSM imaging at low magnification (Fig. 1B) revealed primary and secondary dendrites and the initial segment of the axon. At high magnification single dendritic spines, the smallest neuronal compartments, became apparent (Fig. 1C, arrow). A series of images acquired at different focal planes (Δz = 1.6 μm) demonstrates the sectioning capabilities of the microscope (Fig. 1D-F). For functional imaging of physiological calcium responses, neurons were dialyzed with electrodes containing the calcium indicator Ca-green-1 (300 μM, Molecular Probes). Ca-green is a fluorophore that undergoes a large fluorescence intensity change in response to Ca2+ binding. Intracellular free calcium concentration changes evoked by single action potentials could easily be detected (Fig. 1G).