Calcium-mediated change in neuronal intrinsic excitability in weakly electric fish: biasing mechanisms of homeostatis for those of plasticity

textAlthough the processes used for temporarily storing and manipulating neural information have been extensively studied at the synaptic level far less attention has been given to the underlying cellular and molecular mechanisms that contribute to change in the intrinsic excitability of neurons. More importantly, how do these mechanisms of plasticity integrate with ongoing mechanisms of regulation of neural intrinsic excitability and, in turn, homeostasis of entire neural circuits? In this dissertation I describe the underlying mechanisms that contribute to persistent neural activity and, more globally, sensorimotor adaptation using weakly electric fish as my model system. Weakly electric fish have evolved a behavior adaptation known as the jamming avoidance response (JAR), and it is this adaptation that allows the organism to elevate its own electrical discharge in response to intraspecific interactions and subsequent distortions of the animal’s electric field. The elevation operates over a wide range and in vivo can last tens of hours upon cessation of a jamming stimulus. I demonstrate that the underlying mechanisms of the adaptation are mediated by calcium-dependent signaling in the pacemaker nucleus and that calcium-mediated phosphorylation plays an important role in the regulation of the long-term frequency elevation (LTFE). I demonstrate using an in vitro brain slice preparation from the weakly electric fish, Apteronotus leptorhynchus that the engram of memory formation depends on the cooperativity of calcium-dependent protein kinases and protein phosphatases. In addition, I show that the memory formation (in the form of LTFE) does not depend on the continued flux of calcium, but rather the phosphorylation events downstream of NMDA receptor activation. Moreover, I describe the differences in the expression of protein phosphatases and protein kinases as they relate to species-specific differences in sensorimotor adaptation. It is important to note that this is the first time that the cooperativity between different isoforms of protein kinase C (PKC) have been shown to play a role in graded long-term change in neuronal activity and, in turn, providing the neural basis of species-specific behavior. The neural adaptation of the electromotor system in weakly electric fish provides an excellent model system to study the underlying cellular and molecular events of vertebrate memory formation.Biological Sciences, School o

A roadmap to integrate astrocytes into Systems Neuroscience.

Systems neuroscience is still mainly a neuronal field, despite the plethora of evidence supporting the fact that astrocytes modulate local neural circuits, networks, and complex behaviors. In this article, we sought to identify which types of studies are necessary to establish whether astrocytes, beyond their well-documented homeostatic and metabolic functions, perform computations implementing mathematical algorithms that sub-serve coding and higher-brain functions. First, we reviewed Systems-like studies that include astrocytes in order to identify computational operations that these cells may perform, using Ca2+ transients as their encoding language. The analysis suggests that astrocytes may carry out canonical computations in a time scale of subseconds to seconds in sensory processing, neuromodulation, brain state, memory formation, fear, and complex homeostatic reflexes. Next, we propose a list of actions to gain insight into the outstanding question of which variables are encoded by such computations. The application of statistical analyses based on machine learning, such as dimensionality reduction and decoding in the context of complex behaviors, combined with connectomics of astrocyte-neuronal circuits, is, in our view, fundamental undertakings. We also discuss technical and analytical approaches to study neuronal and astrocytic populations simultaneously, and the inclusion of astrocytes in advanced modeling of neural circuits, as well as in theories currently under exploration such as predictive coding and energy-efficient coding. Clarifying the relationship between astrocytic Ca2+ and brain coding may represent a leap forward toward novel approaches in the study of astrocytes in health and disease

Neurophysiological and Morphological Plasticity in Rat Hippocampus and Medial Prefrontal Cortex Following Trace Fear Conditioning

Pavlovian fear conditioning provides a useful model system for investigating the mechanisms underlying associative learning. In recent years, there has been an increasing interest in trace fear conditioning, which requires conscious awareness of the contingency of CS and US therefore considered as a rodent model of explicit fear. Acquisition of trace fear conditioning requires an intact hippocampus and medial prefrontal cortex (mPFC), but the underlying mechanisms are still unclear. The current set of studies investigated how trace fear conditioning affects neuronal plasticity in both hippocampus and mPFC in adult rats. Trace fear conditioning significantly enhanced both intrinsic excitability and synaptic plasticity (LTP) in hippocampal CA1 neurons. Interestingly, intrinsic excitability and synaptic plasticity were significantly correlated with behavioral performance, suggesting that these changes were learning-specific. The next set of experiments investigated learning-related changes in mPFC. In order to study circuit-specific changes, only neurons that project to the basolateral nucleus of amygdala (BLA) were studied by injecting a retrograde tracer into BLA. Trace fear conditioning significantly enhanced the excitability the layer 5 (L5) projection neurons in the infralimbic (IL) subregion of mPFC whereas it decreased the excitability of L5 projection neurons in the prelimbic (PL) subregion. In both IL and PL, the conditioning effect was time-dependent because it was not observed following a retention (tested 10 days after conditioning). Furthermore, extinction reversed the conditioning effect in both IL and PL, suggesting that these changes are transient and plastic. For comparison, the effects of delay fear conditioning on mPFC neuronal excitability was also studied. These data demonstrated that in adult rats delay fear conditioning significantly enhanced the intrinsic excitability of IL but not PL neurons. However, this conditioning effect was only significant in response to stronger (e.g., larger magnitude) current injections, suggesting that this learning effect was weak. Finally, how trace fear conditioning and extinction modulate dendritic spine density of mPFC-BLA projection neurons was also studied. These data suggest that the spine density is significantly higher in L2/3 neurons than that of L5 neurons, and that extinction facilitates the elimination of spines within L2/3 neurons in both IL and PL. Together these data implicate that both neurophysiological and morphological changes within hippocampus and mPFC are critical for the acquisition and extinction of trace fear conditioning in rats

Neurophysiological characterisation of neurons in the rostral nucleus reuniens in health and disease.

Evidence is mounting for a role of the nucleus reuniens (Re) in higher cognitive function. Despite growing interest, very little is known about the intrinsic neurophysiological properties of Re neurons and, to date, no studies have examined if alterations to Re neurons may contribute to cognitive deficits associated with normal aging or dementia. Work presented chapter 3 provides the first detailed description of the intrinsic electrophysiological properties of rostral Re neurons in young adult (~5 months) C57-Bl/6J mice. This includes a number of findings which are highly atypical for thalamic relay neurons including tonic firing in the theta frequency at rest, a paucity of hyperpolarisation-activated cyclic nucleotide–gated (HCN) mediated currents, and a diversity of responses observed in response to depolarising current injections. Additionally this chapter includes a description of a novel form of intrinsic plasticity which alters the functional output of Re neurons. Chapter 4 investigates whether the intrinsic properties of Re neurons are altered in aged (~15 month) C57-Bl/6J mice as compared to a younger control group (~5 months). The intrinsic properties were remarkably similar across age ranges suggesting that alterations to the intrinsic properties of Re neurons do not contribute to age-related cognitive deficits. Chapter 5 investigates whether alterations to the intrinsic properties of Re neurons occur in the J20 model of amyloidopathy. Alterations to the resting membrane potential (RMP), propensity to rebound fire, and a reduction in action potential (AP) width were observed. This suggests that alterations to the intrinsic properties of Re neurons may contribute to cognitive deficits observed in Alzheimer’s disease (AD). Chapter 6 investigates whether alterations to the intrinsic properties of Re neurons occur in a mouse model (CHMP2Bintron5) of frontotemporal dementia (FTD). Only subtle changes were observed suggesting that alterations to the intrinsic properties of Re neurons does not contribute to cognitive deficits observed in FTD linked to chromosome 3 (FTD-3)

Cortex, countercurrent context, and dimensional integration of lifetime memory

The correlation between relative neocortex size and longevity in mammals encourages a search for a cortical function specifically related to the life-span. A candidate in the domain of permanent and cumulative memory storage is proposed and explored in relation to basic aspects of cortical organization. The pattern of cortico-cortical connectivity between functionally specialized areas and the laminar organization of that connectivity converges on a globally coherent representational space in which contextual embedding of information emerges as an obligatory feature of cortical function. This brings a powerful mode of inductive knowledge within reach of mammalian adaptations, a mode which combines item specificity with classificatory generality. Its neural implementation is proposed to depend on an obligatory interaction between the oppositely directed feedforward and feedback currents of cortical activity, in countercurrent fashion. Direct interaction of the two streams along their cortex-wide local interface supports a scheme of "contextual capture" for information storage responsible for the lifelong cumulative growth of a uniquely cortical form of memory termed "personal history." This approach to cortical function helps elucidate key features of cortical organization as well as cognitive aspects of mammalian life history strategies

Astrocytic modulation of neuronal network oscillations

The synchronization of the neuron’s membrane potential results in the emergence of neuronal oscillations at multiple frequencies that serve distinct physiological functions (e.g. facilitation of synaptic plasticity) and correlate with different behavioural states (e.g. sleep, wakefulness, attention). It has been postulated that at least ten distinct mechanisms are required to cover the large frequency range of neuronal oscillations in the cortex, including variations in the concentration of extracellular neurotransmitters and ions, as well as changes in cellular excitability. However, the mechanism that gears the transition between different oscillatory frequencies is still unknown. Over the past decade, astrocytes have been the focus of much research, mainly due to (1) their close association with synapses forming what is known today as the “tripartite synapse”, which allows them to bidirectionally interact with neurons and modulate synaptic transmission; (2) their syncytium-like activity, as they are electrically coupled via gap junctions and actively communicate through Ca2+ waves; and (3) their ability to regulate neuronal excitability via glutamate uptake and tight control of the extracellular K+ levels via a process termed K+ clearance. In this thesis we hypothesized that astrocytes, in addition to their role as modulators of neuronal excitability, also act as “network managers” that can modulate the overall network oscillatory activity within their spatial domain. To do so, it is proposed that astrocytes fine-tune their K+ clearance capabilities to affect neuronal intrinsic excitability properties and synchronization with other neurons, thus mediating the transitions between neuronal network oscillations at different frequencies. To validate or reject this hypothesis I have investigated the potential role of astrocytes in modulating cortical oscillations at both cellular and network levels, aiming at answering three main research questions: a) what is the impact of alterations in astrocytic K+ clearance mechanisms on cortical networks oscillatory dynamics? b) what specific neuronal properties underlying the generation of neuronal oscillations are affected as a result of impairments in the astrocytic K+ clearance process? and c) what are the bidirectional mechanisms between neurons and astrocytes (i.e. neuromodulators) that specifically affect the K+ clearance process to modulate the network activity output? In the first experimental chapter I used electrophysiological recordings and pharmacological manipulations to dissect the contribution of the different astrocytic K+ clearance mechanisms to the modulation of neuronal network oscillations at multiple frequencies. A key finding was that alterations in membrane properties of layer V pyramidal neurons strongly correlated with the network behaviour following impairments in astrocytic K+ clearance capabilities, depicted as enhanced excitability underlying the amplification of high-frequency oscillations, especially within the beta and gamma range. The second experimental chapter describes a combinatorial approach based on K+-selective microelectrode recordings and optical imaging of K+ ions used to quantitatively determine extracellular K+ changes and to follow the spatiotemporal distribution of K+ ions under both physiological and altered K+ clearance conditions, which affected the K+ clearance rate. The impact of different neuromodulators on astrocytic function is discussed in the third experimental chapter. Using extracellular K+ recordings and Ca2+ imaging I found that some neuromodulators act specifically on astrocytic receptors to affect both K+ clearance mechanisms and Ca2+ signalling, as evidenced by reduced K+ clearance rates and altered evoked Ca2+ signals. Overall, this thesis provides new insights regarding the impact of astrocytic K+ clearance mechanisms on modulating neuronal properties at both cellular and network levels, which in turn imposes alterations on neuronal oscillations that are associated with different behavioural states

Effects of metabolism upon neuronal activity and synaptic performance

Tese de mestrado em Bioquímica, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2013O objectivo desta dissertação é avaliar o efeito do metabolismo (ciclo de alimentação) na atividade neuronal. O trabalho centra-se nas correntes de potássio whole-cell em neurónios piramidais da região CA1 do hipocampo de ratos wistar, particularmente aquelas mediadas pelo canal Kv1.3. Dois objectivos foram estabelecidos: (1) avaliar se a expressão de Kv1.3 e respectiva corrente iónica são afectadas pelo metabolismo e (2) avaliar se o eixo dorsal-ventral do hipocampo é homogéneo no que toca à expressão de Kv1.3 e à sua corrente iónica. Para este efeito, foram usadas abordagens de electrofisiologia e de biologia molecular. Resultados surpreendentes revelam que os neurónios de ratos em período pós-prandial têm diferentes propriedades biofísicas (dependência da voltagem mais despolarizada e cinética mais lenta) e farmacológicas (maior contribuição de correntes sensíveis à margatoxina, possivelmente com dependência de voltagem alterada) em relação a células de ratos em jejum. Propõe-se a existência de um sistema de regulação complexo, de acordo com o ciclo de alimentação, nos neurónios em estudo. Esse sistema envolveria modificações nas populações de canais iónicos que são expressas na membrana neuronal (nomeadamente do Kv1.3, que a margatoxina inibe seletivamente), bem como vias metabólicas capazes de alterar as propriedades biofísicas destes canais (por exemplo alterando a sua fosforilação constitutiva). Mais, os resultados apresentados indicam que o hipocampo dorsal é mais afectado pelo condicionamento metabólico do que o polo ventral, sugerindo que as funções associadas às duas porções do hipocampo são afectas de forma diferente com o ciclo de alimentação. Estes resultados podem dar suporte neuronal para a noção intuitiva de que a consolidação da memória é afetada pelo ciclo de alimentação. Mostra-se ainda que os neurónios piramidais da região CA1 têm características biofísicas e farmacológicas irregulares ao longo do dorso-eixo ventral do hipocampo (as células dorsais têm uma cinética mais lenta, maior densidade de corrente e maior sensibilidade para a margatoxina), associadas a uma expressão diferencial do canal Kv1.3 (maior densidade na parte dorsal). Estes resultados vêm dar mais profundidade a evidências recentes que desafiam a premissa clássica de que os neurónios piramidais de CA1 são uma população electrofisiologicamente homogénea ao longo do eixo longitudinal do hipocampo. Concluindo, as nossas observações sugerem fortemente que a fisiologia dos canais iónicos, bem como a expressão de proteínas no neurónio, alteram-se durante os ciclos metabólicos de jejum/pós-prandial, inclusive ao longo do eixo dorsal-ventral do hipocampo. Fica assim em causa a ideia do cérebro ser uma entidade insensível a variações metabólicas.This study aims to assess the effect of the feeding cycle onto neuronal performance. The work focused on whole-cell K+ currents in pyramidal neurons from CA1 region of wistar rats’ hippocampus, particularly those underlined by Kv1.3 channels. Two goals were established: (1) to assess if Kv1.3 expression and associated ionic currents are affected by metabolism and (2) to evaluate if the dorsal-ventral axis of the hippocampus is homogeneous regarding the Kv1.3 expression patterns and currents. For this purpose, electrophysiology and molecular biology approaches were used. Striking results reveal that fed rats’ neurons have different biophysical (more depolarized voltage-dependence and slower kinetics) and pharmacological (larger margatoxinsensitive currents, possibly with altered voltage-dependence) properties than fasting rats’ cells. It is proposed that a complex regulatory system according to the feeding cycle exists in these neurons. This system would involve modifications in ionic channels populations expressed in the neuronal membrane (namely Kv1.3, that margatoxin selectively inhibits) as well as metabolic pathways capable of altering the biophysical properties of these channels (for example by enhancing their constitutive phosphorylation). Furthermore, the dorsal hippocampus was more extensively affected by metabolic conditioning than the ventral pole, suggesting that functions associated with the two portions of the hippocampus are differently affected by the feeding cycle. These results may give neuronal backing for the intuitive notion of memory consolidation being affected by the feeding cycle. Additionally, CA1 pyramidal neurons had uneven biophysical and pharmacological profiles throughout out the length of the hippocampus - dorsal cells have slower kinetics, larger current density and larger sensitivity to margatoxin. Such uneven features are associated with a differential expression of Kv1.3 channel (higher density in the dorsal portion) throughout the dorsal-ventral axis of the hippocampus. These impressive results give further depth to recent evidence challenging the classical assumption that CA1 pyramidal neurones are an electrophysiologically homogenous population along the longitudinal hippocampal axis. All together, our observations strongly suggest that ion-channel physiology, as well as neural protein expression, change during fast-fed metabolic cycles and within the dorsal-ventral axis of the hippocampus, challenging the idea of a metabolically sealed brain