Dissertations / Theses on the topic 'Layer 5 pyramidal neuron'

Parkinson’s disease (PD) is one of the most prevalent movement disorders in the world. A clinical hallmark of PD is the appearance of proteinaceous Lewy Bodies throughout the brain that are predominantly formed from aggregation of the presynaptic protein alpha-Synuclein (αSyn). Increasing evidence, however, suggests that the soluble annular αSyn oligomers, formed during early stages of aggregation, are more toxic and pathologically relevant than the larger fibrils which form at later stages of aggregation. The underlying mechanism(s) through which αSyn oligomers exert their toxicity is still largely unknown. This thesis investigates how the toxic nature of αSyn oligomers may affect the electrophysiological properties of neurons. A population of soluble oligomers, termed mOligomers, were isolated from the early stages of in vitro aggregation. In addition, a separate oligomeric species was recovered from the fragmentation of large fibrils; termed fOligomers. Structural characterisation of these two species revealed them to be similar in size and ring-like in shape but showed subtle differences in their secondary structure. Purified, oligomeric αSyn was injected directly into the somata of thick-tufted layer 5 pyramidal neurons in mouse neocortical brain slices during whole-cell patch clamp recording and compared to the effects of equivalent concentrations of αSyn monomer. Using a combined experimental and modelling approach, a wide range of neuronal parameters were extracted and demonstrated oligomer-specific changes in neuronal electrophysiology that were time dependent. Perfusion with αSyn oligomers markedly reduced input resistance, enhanced the current required to trigger an action potential and reduced the firing rate illustrating a reduction in excitability that has the potential to impact both neural circuitry and cognitive output.

The actions of many neuromodulators induce changes in synaptic transmission and membrane excitability, and many of these effects are well documented in neurons across the CNS. Adenosine acts as a powerful modulator across the CNS and while its actions have been characterised in some neurons in the neocortex, its effects on excitatory transmission in layer 5 remain unstudied. Adenosine has been implicated in the modulation of spontaneous activity generated in the layer 5 excitatory network, thus understanding its actions in this area are of substantial importance. This study used a combined approach of paired intracellular recordings and quantitative modelling to investigate the actions of adenosine on thick-tufted layer 5 pyramidal neurons in the rat somatosensory cortex. Adenosine was found to powerfully suppress synaptic transmission between these neurons and the changes in synaptic dynamics could be precisely captured as a change only in probability of release in a simple phenomenological model. Recordings conducted at three post-natal ages provide evidence that an increased tone of endogenous adenosine is responsible for the previously described developmental shift in short-term dynamics and reliability of this synapse. The data illustrates both that this endogenous activation of A1 receptors is highly heterogeneous, with variation between neighbouring synapses, and that it plays a significant role in EPSP parameters observed at mature connections. An investigation into adenosine's post-synaptic actions using an approach that measures the neurons' I-V response to naturalistic current inputs demonstrates how adenosine's actions on membrane excitability translate to a strong suppression of spiking. Simultaneous dendritic and somatic recordings demonstrate that this effect is enhanced when current is injected from the dendrite and that back-propagating bursts of action potentials are selectively suppressed by adenosine. As a whole the work illustrates that the effects of adenosine can be well captured by mathematically tractable quantitative models.

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.

Pyramidal cells are the principal excitatory neurons in the cerebral cortex and those in layer 5 (L5) form its primary output. Tufted L5 pyramidal cells present a complex morphology, with non-uniform distributions of active membrane conductances. Their dendritic tree receives thousands of synaptic inputs from local circuits as well as long range inputs from other cortical regions and thalamic nuclei. Hence, the timing of their synaptic inputs are likely to span a wide range of temporal scales, raising the question of how an individual L5 pyramidal cell combines and transforms such temporally and spatially diverse signals. The integrative properties of pyramidal neurons have been extensively studied in vitro and several models have been suggested for the computations performed by these cells. However, cortical pyramidal cells in vivo are constantly bombarded by asynchronous synaptic input, re ecting the activity of the network in which they are embedded. Little is known about how the resulting background activity interacts with nonlinear dendritic properties. I have used experimentally-constrained models of L5 pyramidal neurons to explore synaptic integration under a range of di erent conditions including those measured in vivo. The major result from this study is that background synaptic activity can profoundly alter the integrative properties of pyramidal cells, by activating a distributed NMDA receptor conductance. This distributed nonlinear conductance lowers the threshold for dendritic spikes generation, extends the spikes duration and increases the probability of additional regenerative events occurring in neighbouring branches. Simulations with mixed excitatory/inhibitory background also suggest that dendritic inhibition may be speci cally tuned to regulate this powerful re-generative mechanism. My results suggest a new role for NMDA receptors. During the network activity experienced by pyramidal neurons in vivo, the distributed NMDA conductance may enable pyramidal cells to integrate synaptic input over extended spatio-temporal scales.

Both neuroinflammation, and an increase in microglial cells, have been associated with Autism Spectrum Disorder (ASD) through observation in human subjects as well as in mouse models. A mother having an infection early in pregnancy increases the chances for autism in her child. (Atladottir et al., 2012). This process is known as Maternal Immune Activation (MIA), and the proposed mechanism is that inflammatory signals cross from the mother to child; then in response to increased pro-inflammatory cytokines, microglia within the brain are activated to combat the infection. Microglia are essential to healthy synaptogenesis and neuronal growth, and a change in their signaling early in development has been shown to alter behavior in mouse models that replicate MIA. We use microglial depletion as a therapy to counteract the potentially harmful pro-inflammatory response in the developing mouse brain. Four experimental groups - control, MIA, microglial depleted, and a therapy group (MIA plus microglial depletion)- were run through a comprehensive series of behavioral and electrophysiological assessments. Layer 5 pyramidal cells (L5PNs) were targeted for recording in medial frontal cortex – a mouse cortical area important for cognition and social behavior. L5PNs are a heterogeneous population with cortical and subcortical targeting. Subcortical targeting neurons are thick tufted morphologically, and have an intrinsically bursting spike pattern. Analysis of the intrinsically bursting neurons revealed significant differences between the maternal inflammation and the microglial depletion groups across multiple physiological properties. Therefore, the therapy group had electrophysiological characteristics more consistent with the microglial depleted model than the autism model.

碩士
國立臺灣大學
生命科學系
104
It has been shown that inhibition or lesion of the rostral agranular insular cortex (RAIC) results in analgesia, it suggests that RAIC tonically produces hyperalgesia signal. RAIC is a cortical area where nociceptive output originates, and it has been reported to activate in chronic pain perception. It’s believed that chronic pain is associated with the long-term change in synaptic plasticity. Moreover, the imbalance of excitatory and inhibitory (E/I) synaptic signaling in neural circuits is responsible to modulate synaptic plasticity in certain behavior disorders. In our lab, previous study had reported that the induction of chronic pain induced differential activation in pyramidal cells and GABAergic neurons in RAIC. We propose here that E/I imbalance in RAIC may contribute to the increased cortical output of nociceptive signal in chronic pain. To test this possibility, we compared synaptic transmission of layers 2/3 (L2/3) inputs onto layer 5 (L5) pyramidal cells (PC), which are the descending projection neurons, and onto local GABAergic interneurons (IntN) in RAIC. We performed dual-patch recording from a paired IntN-PC in layer 5, and elicited EPSC by putting an electrode in layer 2/3. We found functional connectivity in 34.2% of all recorded IntN-PC pairs. There was no significant difference in data sampled from IntN-PC pairs with and without functional connectivity, and all data were pooled. Our data showed no significant difference in paring-pulse ratio between transmission at L2/3-PC synapses and at L2/3-IntN synapses. L2/3-IntN seemed to have higher releasing probability than L2/3-PC synapse in quantum study. The ratio of NMDA and non-NMDA EPSCs component was larger at L2/3-PC synapses than at L2/3-IntN synapses. Furthermore, the rising and decay of EPSCs were much faster at L2/3-IntN synapse than at L2/3-PC synapse. We further examined the modulation of pERK on IntN-PC pairs by applying PKC activator Phorbol 12,13- diacetate (PDA). PDA enhanced the postsynaptic currents at L2/3-PC synapses and L2/3- IntN synapses. The further issue of chronic pain model is under studying.

碩士
國立臺灣大學
生命科學系
105
It has been well demonstrated that the change of synaptic efficacy in neurocircuit of brain pain matrix is a cellular substrate for behavior hypersensitivity in animals with chronic pain. While most of previous studies focus on transmission at synapses between nociceptive inputs and principal neurons, the role of local GABAergic interneurons (IntNs) receives less attention. I address this issue by using the acid-induced muscle pain animal model (AIMP model) in mice and focusing on the rostral agranular insular cortex (RAIC). The RAIC is an important component of brain pain matrix as this cortical area is shown to tonically produce hyperalgesia signal and is a cortical area where nociceptive output originates. We propose that repeated acid saline injection may trigger a plastic change in synaptic efficacy of GABAergic IntNs onto pyramidal neurons (PNs) and cause an excitatory/inhibitory imbalance in neurocircuit in RAIC, which in turn alters cortical output of nociceptive signal in chronic pain. To test this possibility, dual-patch recording from a pair of IntN-PN in layer 5 was initially used to record unitary inhibitory postsynaptic current (IPSC) in previous experiments of our lab, and found that only 30% of all recorded IntN-PN pairs showed functional connectivity. To increase successful rate, here I employ optogenetic method to selectively active GABAergic IntNs. I injected a cre-dependent AAV that carries eYFP and channelrhodopsin2 sequences into RAIC in transgenic mice, in which the promoter of vesicular-GABA-transporter controls expression of cre recombinase. The animals were killed 2-3 weeks after AAV injection for brain slice preparation and whole-cell patch recording was made from PNs. Illuminating the slice with a single blue-light pulse (2 ms) evoked inhibitory postsynaptic current (IPSC) in PNs that was blocked by 20 uM bicuculline, a GABAA receptor antagonist. The paired-pulse ratio of the IPSC significantly reduced from 0.66 ± 0.10 (n = 13) in control mice to 0.37 ± 0.03 (n = 12) in muscle pain mice (P < 0.05; Mann-Whitney Test); the quantal size of the IPSC was significantly increased from 12.88 ± 1.22 pA (n = 13) in control mice to 18.82 ± 1.91 (n = 12) pA in muscle pain mice (P < 0.05; Mann-Whitney Test). These results show potential changes in synaptic function of GABAergic IntNs onto PNs in RAIC in chronic pain condition.

The anterior cingulate cortex (ACC) can influence emotional and motivational states in primates by its dense connections with many neocortical and subcortical regions. Pyramidal neurons serve as the basic building blocks of these neocortical circuits, which have been extensively studied in other brain regions, but their morphological and electrophysiological properties in the primate ACC are not well understood. In this study, we used whole-cell patch clamp and high-resolution laser scanning confocal microscopy to reveal the general electrophysiological properties and detailed morphological features of layer 2 and 3 pyramidal neurons in ACC (area 24/32) of the rhesus monkey. Neurons from both layers had similar passive membrane properties and action potential properties. Morphologically, dendrites of layer 3 ACC neurons were more complex than those of layer 2 neurons, by having dendrites with longer total dendritic lengths, more branch points and dendritic segments, spanning larger convex hull volumes. This difference in total dendritic morphology was mainly due to the apical dendrites. In contrast, the basal dendrites displayed mostly similar features between the two groups of neurons. However, while apical dendrites extend to the same layer (layer 1), the basal dendrites of layer 3 extended into deeper layers than layer 2 because of the difference in soma-pia distance. Thus, basal dendrites of the two groups of neurons receive different laminar inputs. Analysis of spines showed that more spines were found in neurons of layer 3 apical dendritic arbors than layer 2 neurons. However, the apical spine densities were similar between neurons in the two layers. Thus, while higher spine number suggests that layer 3 neurons receive more excitatory input than layer 2 neurons, the similar spine density suggests similar spatial and temporal summation of these inputs. The combined effects of increased number of excitatory input and higher dendritic complexity in layer 3 than in layer 2 ACC neurons suggest the additional information received by layer 3 neurons, especially in the apical dendrites, might undergo more complex integration.

Rett Syndrome (RTT) is a neurodevelopmental disorder primarily caused by mutations in the X-linked gene methyl-CpG-binding protein 2 (MECP2). The mosaic brain environment in heterozygous (MECP2+/-) females consists of both MeCP2-wildtype (MeCP2+) and Mecp2-mutant (MeCP2-) neurons. To separate possible cell autonomous and cell non-autonomous effects three-dimensional morphological analysis was performed on individually genotyped layer V pyramidal neurons in the primary motor cortex of heterozygous (Mecp2+/-) and wild-type (Mecp2+/+) mature female mice (>8 months old) from the Mecp2tm1.1Jae line. Mecp2+/+ neurons and Mecp2+ were found to be indistinguishable while Mecp2- neurons have significantly reduced basal dendritic length (p<0.05), predominantly in the region 70-130 μm from the cell body, culminating in a total reduction of 15%. Mecp2- neurons have three (17%) fewer total branch points, lost specifically at the second and third branch orders. Thus the reduced total dendritic length in Mecp2- neurons is a result of fewer higher-order branches. Soma and nuclear areas of 30 Mecp2+/- female mice (5-21 months) with X chromosome inactivation (XCI) ratios ranging from 12% to 56% were analyzed. On average Mecp2- somata and nuclei were 15% and 13% smaller than Mecp2+ neurons respectively. The variation observed in the soma and nuclear sizes of Mecp2- neurons was not due to age, but was found to be correlated with the XCI ratio. Animals with a balanced XCI ratio (approximately 50% Mecp2-) were found to have Mecp2- neurons with a less severe cellular phenotype (11-17% smaller than Mecp2+). Animals with a highly skewed XCI ratio favouring expression of the wild-type allele (less than 30% Mecp2-) were found to have a more severe Mecp2- cellular phenotype (17-22% smaller than Mecp2+). These data support indicate that mutations in Mecp2 exert both cell autonomous and cell non- autonomous effects on neuronal morphology.
Graduate

Affective disorders represent one of the greatest global burdens of disease. Work in patients with affective disorders demonstrates that serotonin (5-HT) signaling within the prefrontal cortex, particularly at the level of the 5-HT receptors, plays an integral role in both the pathology and treatment of these diseases. Surprisingly, the characterization of the prefrontal 5-HT receptors under both healthy and pathological conditions remains incomplete. The technique of whole cell electrophysiological recording provides an unparalleled tool for investigating the functional effects of these 5-HT receptors on neurons in acute prefrontal cortical slices. The objectives of my thesis were to delve deeper into the 5-HT receptor subtypes that modulate the prefrontal cortex in the healthy control rodents and to examine how this modulation was disrupted in a rodent model of affective disorders. In work from healthy control rodents, I examined two prefrontal 5-HT receptor-mediated currents. I show for the first time the presence of the 5-HT1A receptor during the early postnatal period, a critical developmental window during which this receptor programs adult anxiety behaviors. In adulthood, I characterized an inhibitory current mediated by the 5-ht5A receptor; findings that will permit the classification of this receptor within the 5-HT receptor family. Collectively, this investigation of functional early 5-HT1A receptors and adult 5-ht5A receptors offers a novel conceptual framework for understanding 5-HT receptor modulation of the healthy prefrontal cortex. To model vulnerability to affective disorder in the rodent, I used the early stress of maternal separation. In early stress rodents, I observed a marked increase in 5-HT1A receptor currents during the early postnatal period, the critical time window for the programming of anxiety. By comparison, in adulthood I found that rodents exposed to early stress displayed increased 5-HT2A receptor currents. These findings provide novel insight into the developmental and long-lasting pathology underlying early stress, indicating that the early prefrontal 5-HT1A receptor and adult prefrontal 5-HT2A receptors as a potential therapeutic target in treatment of affective disorders At a fundamental level, the findings provided herein offer critical insight into the cellular mechanisms underlying affective disorders, one of the most debilitating and costly diseases worldwide.