Thesis (M.S.)--University of Michigan, 1992.

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 198-229).

Orientadores: Elenice Aparecida de Moraes Ferrari, Claudio Antonio Barbosa de Toledo / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-10T10:38:58Z (GMT). No. of bitstreams: 1 Silva_MariaIsabel_M.pdf: 1381550 bytes, checksum: 9cb336aef794086ff363f071f8040818 (MD5) Previous issue date: 2007 / Resumo: O hipocampo exerce papel fundamental no processamento de aprendizagem e memória espaciais. Comparações das características funcionais, anatômicas e neuroquímicas do hipocampo são favorecidas por evidência oriunda de estudo sobre aprendizagem especial em mamíferos e aves. O objetivo do presente estudo foi analisar a marcação imunohistoquímica de células nNOS- positivas no hipocampo de pombos (C. lívia) após diferentes duração do treino em aprendizagem especial. Foram analisados grupos de animais não treinados (MAN), treinados em 1 sessão (EXP1), treinados em 5 sessões (EXP5), exposto à arena em 1 sessão (CONT1) ou em 5 sessões (CONT5). As sessões foram realizadas numa arena onde havia quatro comedouros, um dos quais com alimento. Em cada sessão, com seis tentativas, registrou-se a latência (seg) e a assertividade da escolha de um comedouro. Após os testes comportamentais, usou-se imunoistoquímica para a análise da marcação de células nNOS-positiva no hipocampo dorsal e ventral. O grupo EXP5 teve diminuição da latência de escolha ('F IND. 4,28¿= 23,74; p < 0,001) e aumento das respostas corretas ('F IND. 4,35¿= 8,66; p < 0,001) em função do treino. A marcação das células nNOS-positivas no hipocampo foi significativamente maior no hipocampo dorsal dos animais EXP5 em comparação com o hipocampo ventral ('F IND. 4,22¿= 104,79; p<0,001) e com os demais grupos ('F IND. 4,22¿= 10,17; p < 0,001). O aumento da imunorratividade de células nNOS- positivas no hipocampo dorsal de pombos após a aprendizagem da localização do comedouro correto sugere o envolvimento desta região e de processos mediados pro transmissão glutamatérgica nesse processo de aprendizagem e memória em pombos / Abstract: The hippocampus has fundamental role in spatial learning and memory processes. Functional and neurochemical analysis of the hippocampus are favored by evidence on spatial learning in mammals and birds. The present study examined the immunohistochemical expression of nNOS-positive cells in the hippocampus of pigeons (C. livia) after training in food location task. Animals were trained in one (EXP1) or five (EXP5) sessions or had one (CONT1) or five sessions (CONT5) of exposure to the experimental arena. The six trials sessions were conducted daily in one arena with 4 food bowls, one of which had food. Latency and accuracy of choise recorded. After behavioral tests, nNOS immunoractivity in hippocampal cells was analyzed. EXP 5 showed reduction imunoreactivity in hippocampal cells was analysed. EXP5 showed reduction in latency of choise ('F IND. 4,28¿= 23,74; p < 0,001) and increassis in correct choise ('F IND. 4,35¿= 8,66; p < 0,001) as function of the training. The expression of nNOS- positive cells was significantily higher in the dorsal hippocampus of EXP5 group as compared with the ventral hippocampus ('F IND. 4,22¿= 104,79; p < 0,001) and the other groups ('F IND. 4,28¿= 10,17; p < 0,001). The increases of nNOS immunoreactive neurons after learning of the food location suggest that nNOS is involved in processes of spatial learning and memory that are mediated by the dorsal hippocampus of pigeons / Mestrado / Fisiologia / Mestre em Biologia Funcional e Molecular

Orientador: Elenice Aparecida de Moraes Ferrari / Disertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-13T18:30:13Z (GMT). No. of bitstreams: 1 Dias_ElayneVieira_M.pdf: 1520883 bytes, checksum: 05673dcedfdc35b300eb07144edbd5bb (MD5) Previous issue date: 2008 / Resumo: O condicionamento clássico aversivo é utilizado para investigar os mecanismos celulares e moleculares na formação da memória em diferentes espécies de animais. Estes envolvem processos sinápticos que desencadeiam mecanismos de sinalização intracelular com ativação de diferentes quinases em momentos específicos. A ativação da PKC é um dos mecanismos moleculares da plasticidade sináptica subjacente à formação de memória. O presente trabalho investigou o envolvimento da PKCá/âIl no condicionamento clássico aversivo em pombos. No Experimento 1, o inibidor da PKC, calfostina C foi administrado i.c.v. em um grupo de pombos (GCdCa, n=6; 5ml de solução 60mg/ml, DMSO 2%), 1h antes do condicionamento. Outro grupo recebeu veículo (GCdVe, n=5; DMSO 2% em salina). A sessão de condicionamento teve 20 min de duração e 3 pareamentos som-choque (treino). O teste ao contexto ocorreu 24h após o treino. O Experimento 2 usou grupos de pombos expostos ao contexto experimental (GCC), som e choque não pareados (GCR) ou som-choque pareados (GCd) para investigar a ativação da PKCá/âII no hipocampo 2h após o treino, por meio de Western blot. No Experimento 3, grupos de pombos não treinados (GC, n=6) ou sacrificados em diferentes tempos após o treino - G1min (n=6), G1h (n=6), G2h (n=6) e G24h (n=6) - foram utilizados para investigar o curso temporal da ativação da PKCá/âII e da fosforilação do substrato da PKC, GAP-43, no hipocampo. Todas as sessões foram gravadas em vídeo para posterior análise dos dados comportamentais. No Experimento 1 o GCdCa teve menor expressão da resposta condicionada de congelamento (freezing) ao contexto em comparação ao GCdVe (p<0,05), indicando que a administração da calfostina C prejudicou a memória aversiva contextual. Não ocorreram diferenças significativas na ativação da PKC á/âII entre os diferentes grupos (Experimentos 2 e 3;p>0,05), mas houve maior imuno-marcação da GAP-43 fosforilada no G1min quando comparado ao GC (Experimento 3; p<0,05). Esses dados indicam o envolvimento da PKC em mecanismos de aprendizagem e memória aversiva em pombos, e sugerem que outras isoformas além da PKCá/âII podem participar desses processos. / Abstract: The classical aversive conditioning is used to investigate cellular and molecular mechanisms of memory formation in different animal species. Those mechanisms involve synaptic processes that trigger intracellular signaling with activation of different kinases at specific time points. The PKC activation is one of the molecular mechanisms of synaptic plasticity underlying memory. This study investigated the involvement of PKCá/âIl in classical aversive conditioning in pigeons. In Experiment 1, the PKC inhibitor, calphostin C was administered i.c.v. in one group of pigeons (GCdCa, n=6; 5ml solution 60mg/ml, DMSO 2%), 1h before the conditioning. Another group received vehicle (GCdVe, n=5; DMSO 2% in saline). The session of conditioning had 20 min duration and 3 tone-shock pairings (training). The test to the context occurred 24h after training. Experiment 2 investigated with Western blot analysis the PKCá/âII activation in the hippocampus 2h after the training in groups of pigeons that were exposed to unpaired (GCR) or paired (GCd) tone-shock presentations or to the experimental context only (GCC). In Experiment 3, groups of pigeons naive (GC, n=6) or sacrificed at different times after the training - G1min (n=6), G1h (n=6), G2h (n=6) and G24h (n=6) - were used to investigate the time course of the PKCá/âII activation and phosphorylation of PKC substrate, GAP-43, in the hippocampus. All sessions were video recorded for analysis of behavioral data. In Experiment 1 GcdCa had lower expression of conditioned freezing response to the context in comparison to GCdVe (p<0.05), indicating that calphostin C administration impaired contextual aversive memory. No significant differences in the PKCá/âII activation were observed among the groups (Experiments 2 and 3; p>0.05) but the immunolabeling of phosphorylated GAP-43 in G1min was higher as compared to GC (Experiment 3; p<0.05). These data indicate the involvement of PKC in mechanisms of aversive learning and memory in pigeons and suggest that other isoforms besides PKCá/âII may play a role in those processes. / Mestrado / Fisiologia / Mestre em Biologia Funcional e Molecular

The mechanisms underlying the generation and spread of seizure activity have remained elusive despite a considerable research effort over the last two decades. Most of this work has concentrated on the characteristics of neuronal excitability and burst discharge at the single cell level. These studies have provided some understanding of the possible abnormalities of neurons within an epileptic focus, but little direct insight into the factors responsible for the striking synchronization of action potentials during interictal discharge or in the spread of synchronous activity across apparently normal brain tissue. Although synaptic activation probably plays a role in the generation of seizure activity, recent evidence indicates that seizure-like discharge can occur during chemical blockade of synaptic transmission (Jefferys and Haas 1982; Taylor and Dudek 1982). This rather surprising result emphasizes the importance of considering non-synaptic mechanisms for both the synchronization and spread of abnormal neuronal activity in the central nervous system. One important non-synaptic mechanism to consider is ephaptic interactions. This term refers to the direct electrical influence of extracellular field potentials on neuronal excitability. It is possible that ephaptic interactions, generated during seizure activity, simultaneously depolarize an entire population of neurons leading to both recruitment and synchronization of action potential discharge. This thesis investigates ephaptic interactions during evoked potentials in the hippocampal formation. The hippocampus is one of the most seizure-prone regions of the brain and its anatomical structure is ideal for the generation of field effects. Evoked potentials were used as "models" of synchronous neuronal discharge since they are more reproducible, easier to control, and better understood than seizure activity. This initial investigation of ephaptic interactions lays the foundation for further studies involving the complexities of epileptic activity. The first phase of this project examined the spatial characteristics of field potentials evoked in the hippocampus and the dentate gyrus. Current source density (CSD) analysis and voltage gradient determinations obtained from these fields were used to characterize the pattern of current flow within the neuropil and to predict the polarity and relative intensity of ephaptic influences on neuronal excitability. The detailed characteristics of extracellular voltage gradients varied between CAl and the dentate gyrus, and also between anti- and orthodromic responses. In general, voltage gradients during the positive components of a somatic population spike predicted ephaptic hyperpolarization of the neuronal population, whereas gradients observed during the negative component predicted depolarization. They were often an order of magnitude greater than the smallest gradient known to influence granule cell activity. An exception to this rule was the minimal gradient observed during the negative component of the dentate response. In the second phase of the study, extracellular voltage gradients were experimentally applied to the dentate gyrus to determine the sensitivity of granule cells to ephaptic interactions. The magnitude of the applied gradients were in the range observed during the evoked potentials studied in the first phase. These experiments demonstrated a remarkable sensitivity of granule cells to the applied fields. The fields could alter the population spike from near minimal to near maximal. Surprisingly, even antidromic potentials were influenced by the gradients. On the other hand, the EPSP phase of the population spike was not influenced. These findings established that extracellular currents can influence the excitability within a neuronal population without altering synaptic drive. The final phase of the project investigated the transmembrane potential (TMP) of pyramidal and granule cells during applied fields and evoked potentials. The TMP was calculated by subtracting the extracellular from the intracellular response. This potential ultimately determines the voltage dependent behavior of a neuron and gives a direct measure of any ephaptic interactions. In order to measure the intracellular influences of applied fields, the TMP was monitored while the impaled cell was exposed to extracellular voltage gradients spanning the same range as used in phase two of the project. The TMP shifted by as much as plus or minus 5 mV, depending on the amplitude and polarity of the gradient. This large shift in TMP accounts for the observed influence of the applied field potentials, and suggests that the voltage gradients associated with evoked potentials should also have a marked effect on the TMP. A depolarizing wave of the TMP occurred during the negative component of anti- and orthodromic CA1 responses. This depolarization was capable of initiating action potentials, and decreased the latency to discharge during orthodromic responses. During epileptiform discharge, a similar depolarizing wave was associated with each negative component of the burst. These depolarizations recruit and synchronize neuronal discharge by simultaneously increasing the excitability within an entire population of cells. These data support the hypothesis that ephaptic interactions in the hippocampal formation influence the pattern of cell discharge during evoked potentials. It is postulated that similar ephaptic interactions may contribute to recruitment and synchronization during seizure activity. / Medicine, Faculty of / Cellular and Physiological Sciences, Department of / Graduate

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

Simple circuits perform simple tasks. Complex circuits can perform more complicated tasks. This is true for artificial circuits and for brain circuits. As is known from artificial networks, a complexity that makes circuits substantially more powerful is distributing learning across multiple layers. In fact, most brain circuits in vertebrate systems are multi-layer circuits (but for few that perform simple reflexes) in which learning is distributed across layers. Despite the crucial contribution of learning in middle layer neurons to the output of the circuits they are embedded in, there is little understanding of the principles defining this contribution. A very common feature in brain circuits is that middle layer neurons generate two types of signals, known as spikes. These middle layer neurons commonly have long dendrites where they generate dendritic spikes. As well, like most neurons, they generate axonal spikes near the cell body. Neurons exhibiting these two spike types include pyramidal cells in the neo-cortex and the hippocampus, the Purkinje cells in the cerebellum and many more. In this thesis I study another circuit that contains middle layer neurons, the electrosensory lateral lobe (ELL) of the mormyrid fish. The ELL is a tractable brain circuit in which the middle layer neurons generate dendritic and axonal spikes. In this thesis I show that these spike types are not two different expressions of the same inputs. Rather, they have a symbiotic relationship. Instead of all inputs triggering both spikes, some inputs can selectively drive dendritic spikes. The dendritic spikes in return modify the synaptic strength of another set of inputs. The modified inputs are then transmitted to downstream neurons via the axonal spikes, which contributes a desired signal to the output of the circuits. Effectively there is a separation of learning and signaling in the middle layer neurons through the two spike types. Having two types of spikes in the same neuron doing different computations enormously expands the computational power of the neuron. But, being in the same neuron means the separation of function is constrained and needs to be supported by biophysical principles. I have thus built a biophysical model to understand the biophysical principles underlying the separation of function. I show that in the middle layer neurons of the ELL, the axonal spikes are strongly reduced in amplitude as they backpropagate to the apical dendrites, yet they remain crucial in driving dendritic spikes. Critically, modulation of inhibitory inputs can selectively dial up or down the ability of the backpropagating axonal spikes to drive dendritic spikes. Thus, a set of inhibitory modulating inputs can selectively modulate dendritic spikes. Having learning in different layers contributing to the outcome of the circuit, naturally leads to asking how the work is divided across layers and neuron types within the circuit. In this thesis I answer this question in the context of the outcome of the ELL circuit. Finally, another signature of a complex circuit is the ability to integrate many different inputs, usually in middle layer neurons, to generate sophisticated outputs. A goal for scientists studying systems neuroscience is to understand how this integration works. In this thesis I provide a coherent model of a learning behavior called vestibulo occular reflex (VOR) adaptation, that depends on the integration of separate inputs to yield a learned behavior. The VOR is a simple reflex generated in the brain stem. Inputs from the brain stem are also sent to an area in the cerebellar cortex called the flocculus. The flocculus also receives another set of inputs that generate a different behavior, called smooth-pursuit. The integration of VOR inputs with smooth-pursuit inputs in the flocculus generate VOR adaptation. Understanding complex circuits is one of the greatest challenges for today's neuroscientists. In this thesis I tackle two such circuits and hope that through a better understandings of these circuits we gain principles that apply to other circuits and thereby advance our understanding of the brain.

Temporal lobe epilepsy (TLE) is among the most common forms of epilepsy in adults. A significant proportion of patients experience drug-resistant seizures associated with hippocampal sclerosis (HS), in which there is extensive cell loss in the hippocampal cornu ammonis 1 (CA1) and cornu ammonis 3 (CA3) subfields. The dentate gyrus (DG) and cornu ammonis 2 (CA2) subfield are more resilient to neurodegeneration, and a prior report found that CA2 neurons in tissue from TLE patients show interictal-like firing and receive aberrant perisomatic excitatory synapses from DG granule cell (GC) mossy fibers (Wittner et al. Brain. 2009;132:3032–3046). Furthermore, findings from a collaborative study in the laboratory of Dr. Helen Scharfman demonstrated that chronic chemogenetic inhibition of CA2 pyramidal neurons (PNs) in vivo significantly reduced the frequency of spontaneous recurring convulsive seizures in epileptic mice. I therefore explored the hypothesis that pathophysiological changes to CA2 PN excitability or synaptic connectivity may be associated with chronic epilepsy by examining CA2 properties in a mouse model of TLE.Pilocarpine-induced status epilepticus in mice leads to a pattern of hippocampal sclerosis-like neurodegeneration and recurring spontaneous seizures, and thus recapitulates key features of TLE. I performed whole-cell electrophysiological recordings from PNs in acute hippocampal slices from pilocarpine (PILO)-treated mice in the chronic phase of epilepsy as well as age-matched controls. In some experiments I used Cre-expressing mouse lines to selectively express a light-activated excitatory channel in CA2 PNs or DG GCs. I also performed immunohistochemistry to examine CA2 interneuron (IN) populations following PILO-induced status epilepticus. I found that in healthy tissue CA2 PNs, like those in CA3, both directly excited other CA2 PNs via a recurrent CA2-CA2 PN circuit and indirectly inhibited other CA2 PNs by recruiting local INs. The CA2 and CA3 subfields also form reciprocal excitatory and feedforward inhibitory circuits. These recurrent and reciprocal circuits constitute an auto-associative network in which INs crucially control CA2/CA3 population excitability. DG GC mossy fibers made direct but relatively weak excitatory synapses onto CA2 PNs. Following PILO-induced status epilepticus, feedforward inhibition is diminished in the DG GC mossy fiber circuit to CA2, in the CA2/CA3 recurrent network, and in the forward-projecting circuit from CA2 PNs to CA1. I found a modest decrease in the density of parvalbumin-immunopositive INs and a profound decrease of cholecystokinin-immunopositive IN density, combined with degradation of the pyramidal neuron-associated perisomatic perineuronal net, which together may contribute to this inhibitory disruption. DG GC mossy fiber excitatory input to CA2 PNs is strengthened, along with CA2 PN excitatory input to CA1 PNs. Finally, in hippocampal slices from PILO-treated mice I found an increase in CA2 PN input resistance and thus elevated intrinsic excitability, leading to a higher firing rate upon direct current injection. The combined effect of these changes may drive the emergence of epileptiform synchronization in the CA2 network and facilitate the propagation of seizure activity from the DG and entorhinal cortex directly to CA1 via the CA2-centered disynaptic (EC LII --> CA2 --> CA1) and alternate trisynaptic circuits (EC LII --> DG --> CA2 --> CA1).

The world is a complex and dynamic place. The incredibly dense and constantly changing information stream with which our senses are bombarded must be decomposed, taken in, and processed by any organism hoping to make enough sense of this world in order to survive to the next moment. For complex behaviors, and in particular a great many of those that we often feel define us as a human species, this dense sensory stream must not just be processed, but the important features of the environment must be further distilled and structured into representations that can then be stored long-term to guide future behavior through the joint processes of Learning and Memory. The primary goal of this thesis is to further our understanding of the neurobiological bases - at the subcellular, circuit, and network level - of learning and memory. The hippocampus, one of the most studied systems in the brain by far, is thought to play a central role in learning and memory. Principal cells in the hippocampus become tuned to environmental features, forming persistent representations of an animal’s environment, but the precise mechanisms by which these representations are formed, used, and maintained remain unresolved. By employing a variety of experimental techniques including in vivo two-photon calcium imaging, extracellular electrophysiology, optogenetics, and chemogenetics in awake, behaving mice, we attempted to characterize the subcellular and circuit determinants of place field representations and to connect them to these cells’ role in spatial learning and memory.

Historically, the role of immune cells in the nervous system was predominantly examined throughthe lens of disease. In recent years, studies have shown that the complex, orchestrated events of immune activity throughout embryonic and postnatal critical periods are crucial for proper nervous system development. While previous studies have suggested limited immune heterogeneity in the adult brain, the diverse roles of the hippocampus in cognition and pathological development would suggest variation of immune cells in this region. Specifically, the hippocampus is known to be a site of adult neurogenesis. However, fundamental traits of immune cells in this region have not been well characterized. In chapter one, I present a summary of literature that discusses what was previously known of immune regulation of adult neurogenesis during health and disease. In chapter two, I compare different reporter lines and marker genes to evaluate responses in various cell types in the neurogenic niche and in other regions of the brain in the context of injury and pharmacological modulation. I discuss preliminary evidence suggesting microglial depletion may result in phenotypic changes in astrocytes throughout the hippocampus. In chapter three, I provide evidence of heterogeneity in myeloid-lineage cells in the hippocampus. I leveraged the highthroughput nature of cell suspension based single cell RNA-sequencing to collect transcriptomes of over 20,000 myeloid lineage cells from murine hippocampi. Using a series of bioinformatic techniques, I was able to computationally dissect different populations within this system and found spatial mapping of one distinct subset specifically localized to the neurogenic niche of the hippocampus. The transcriptomic signature of these cells alongside immunoreactivity to candidate genes, and morphological properties of this population resemble those of reactive microglia associated with the restriction of neurodegenerative diseases. In chapter four, I discuss how the immune landscape of the hippocampus responds to perturbation using a model of Focused Ultrasound mediated Blood-Brain Barrier opening. Subtypes of myeloid lineage cells change in composition and in transcriptomic response. We find distinct, temporally defined transcriptional responses in microglial and macrophage populations, indicating discrete roles for microglia and macrophages in immune activity during the transition from acute to chronic response. Together, these findings point towards diverse properties of microglia in the adult hippocampus.