The Effects of NMDA Subunit Composition on Calcium Influx and Spike Timing-Dependent Plasticity in Striatal Medium Spiny Neurons

Calcium through NMDA receptors (NMDARs) is necessary for the long-term potentiation (LTP) of synaptic strength; however, NMDARs differ in several properties that can influence the amount of calcium influx into the spine. These properties, such as sensitivity to magnesium block and conductance decay kinetics, change the receptor's response to spike timing dependent plasticity (STDP) protocols, and thereby shape synaptic integration and information processing. This study investigates the role of GluN2 subunit differences on spine calcium concentration during several STDP protocols in a model of a striatal medium spiny projection neuron (MSPN). The multi-compartment, multi-channel model exhibits firing frequency, spike width, and latency to first spike similar to current clamp data from mouse dorsal striatum MSPN. We find that NMDAR-mediated calcium is dependent on GluN2 subunit type, action potential timing, duration of somatic depolarization, and number of action potentials. Furthermore, the model demonstrates that in MSPNs, GluN2A and GluN2B control which STDP intervals allow for substantial calcium elevation in spines. The model predicts that blocking GluN2B subunits would modulate the range of intervals that cause long term potentiation. We confirmed this prediction experimentally, demonstrating that blocking GluN2B in the striatum, narrows the range of STDP intervals that cause long term potentiation. This ability of the GluN2 subunit to modulate the shape of the STDP curve could underlie the role that GluN2 subunits play in learning and development

A Kinetic Model of Dopamine- and Calcium-Dependent Striatal Synaptic Plasticity

Corticostriatal synapse plasticity of medium spiny neurons is regulated by glutamate input from the cortex and dopamine input from the substantia nigra. While cortical stimulation alone results in long-term depression (LTD), the combination with dopamine switches LTD to long-term potentiation (LTP), which is known as dopamine-dependent plasticity. LTP is also induced by cortical stimulation in magnesium-free solution, which leads to massive calcium influx through NMDA-type receptors and is regarded as calcium-dependent plasticity. Signaling cascades in the corticostriatal spines are currently under investigation. However, because of the existence of multiple excitatory and inhibitory pathways with loops, the mechanisms regulating the two types of plasticity remain poorly understood. A signaling pathway model of spines that express D1-type dopamine receptors was constructed to analyze the dynamic mechanisms of dopamine- and calcium-dependent plasticity. The model incorporated all major signaling molecules, including dopamine- and cyclic AMP-regulated phosphoprotein with a molecular weight of 32 kDa (DARPP32), as well as AMPA receptor trafficking in the post-synaptic membrane. Simulations with dopamine and calcium inputs reproduced dopamine- and calcium-dependent plasticity. Further in silico experiments revealed that the positive feedback loop consisted of protein kinase A (PKA), protein phosphatase 2A (PP2A), and the phosphorylation site at threonine 75 of DARPP-32 (Thr75) served as the major switch for inducing LTD and LTP. Calcium input modulated this loop through the PP2B (phosphatase 2B)-CK1 (casein kinase 1)-Cdk5 (cyclin-dependent kinase 5)-Thr75 pathway and PP2A, whereas calcium and dopamine input activated the loop via PKA activation by cyclic AMP (cAMP). The positive feedback loop displayed robust bi-stable responses following changes in the reaction parameters. Increased basal dopamine levels disrupted this dopamine-dependent plasticity. The present model elucidated the mechanisms involved in bidirectional regulation of corticostriatal synapses and will allow for further exploration into causes and therapies for dysfunctions such as drug addiction

Non-linear synaptic integration on dendrites of striatal medium-spiny neuron : a computational study

Striatum is the main input nucleus of basal ganglia. Medium-spiny neurons (MSNs), the principal neurons of the striatum, receive convergent excitatory inputs from cortex and thalamus, thus “gate” the information flow to the basal ganglia. The activity of MSNs is further modulated by massive inhibition from their neighboring MSNs as well as from GABAergic interneurons. At corticostriatal synapses in MSNs, a potent and reliable spike timing-dependent plasticity (STDP) can be found. It has been suggested this plasticity follows an “anti-Hebbian” learning rule: pre-synaptic signals preceding post-synaptic action potentials (‘pre-post’ paring) induces LTD while post-synaptic action potentials preceding pre-synaptic signals (‘postpre’ paring) leads to LTP. The long-term potentiation (LTP) relies on NMDAR-mediated calcium influx, while the long-term depression relies on L-type calcium channels and endocannabinoid (eCB) dependent signaling pathways. The sign ofSTDP rule at the corticostriatal synapses appears to be influenced by the presence of GABAergic inputs. In addition to the role of synaptic interactions for modulating and controlling plasticity, synaptic interactions can also give rise to “dendritic plateaus” were found in MSNs. Clustered activation ofspines at distal dendrites, within a short temporal window, can evoke a long-lasting plateau potential in MSNs. It is generally assumed that this supra-linear integration could promote spiking in MSNs, however, it has not been clear how dendritic plateaus are controlled by excitatory and inhibitory inputs in MSNs. In this thesis, using biophysically detailed models of MSNs, we explored: (1) the possible mechanisms of GABA in STDP formation, (2) the roles of different NMDAR subunits in STDP formation, and (3) how dendritic plateaus affect the integration of excitatory and inhibitory inputs in MSNs. We found that in brain slices the GABA tightly controlled the polarity of STDP in MSNs, while blocking GABA could reverse the STDP rule from anti-Hebbian learning to Hebbian. Surprisingly, the model predicted that GABA depolarizes the dendrites during the STDP protocols and such depolarizing effects further change the balance between NMDA-mediated calcium and the calcium influx from L-type calcium channels. In “pre-post” parings, the GABA strength pushes the balance towards L-type calcium, thus promoting LTD formation. In contrast, during “post-pre” parings, the presence of GABA pushes the balance more towards NMDAR-mediated calcium, thus favoring LTP formation. Next, we identified the role of NMDAR subunits in LTP formation. The model predicted that the GluN2B subunit could broaden the timing window of LTP. We confirmed the prediction with experiments. At last, we investigated the functional importance of dendritic plateaus in MSNs. The model predicted that dendritic plateaus could enhance neuron-wide integration of excitatory inputs and promote spiking. In contrast, the impact of dendritic inhibition depends on a particular “spatiotemporal” window: the efficacy of dendritic inhibition could be dramatically increased if it is positioned close to the plateau initiation zone and activated within a specific timing window. Intriguingly, the model predicted that such branch-specific inhibition is not due to shutting of GABAARs, but relies on the Magnesium (Mg2+) block of NMDARs. We verified the mechanism with two-photon uncaging of glutamate and single-photon uncaging of GABA. To conclude, we found GABA tightly controlled the direction of STDP in MSNs through depolarizing effects and could effectively suppress the dendritic plateau in MSNs through an NMDAR Mg2+ block dependent mechanism

Modulation of Spike-Timing Dependent Plasticity: Towards the Inclusion of a Third Factor in Computational Models

In spike-timing dependent plasticity (STDP) change in synaptic strength depends on the timing of pre- vs. postsynaptic spiking activity. Since STDP is in compliance with Hebb’s postulate, it is considered one of the major mechanisms of memory storage and recall. STDP comprises a system of two coincidence detectors with N-methyl-D-aspartate receptor (NMDAR) activation often posited as one of the main components. Numerous studies have unveiled a third component of this coincidence detection system, namely neuromodulation and glia activity shaping STDP. Even though dopaminergic control of STDP has most often been reported, acetylcholine, noradrenaline, nitric oxide (NO), brain-derived neurotrophic factor (BDNF) or gamma-aminobutyric acid (GABA) also has been shown to effectively modulate STDP. Furthermore, it has been demonstrated that astrocytes, via the release or uptake of glutamate, gate STDP expression. At the most fundamental level, the timing properties of STDP are expected to depend on the spatiotemporal dynamics of the underlying signaling pathways. However in most cases, due to technical limitations experiments grant only indirect access to these pathways. Computational models carefully constrained by experiments, allow for a better qualitative understanding of the molecular basis of STDP and its regulation by neuromodulators. Recently, computational models of calcium dynamics and signaling pathway molecules have started to explore STDP emergence in ex and in vivo-like conditions. These models are expected to reproduce better at least part of the complex modulation of STDP as an emergent property of the underlying molecular pathways. Elucidation of the mechanisms underlying STDP modulation and its consequences on network dynamics is of critical importance and will allow better understanding of the major mechanisms of memory storage and recall both in health and disease

Effect of age and unilateral dopamine depletion on striatal NMDA receptor function

Includes bibliographical references

Presynaptic NMDA Receptors and Spike Timing-Dependent Depression at Cortical Synapses

It has recently been discovered that some forms of timing-dependent long-term depression (t-LTD) require presynaptic N-methyl-d-aspartate (NMDA) receptors. In this review, we discuss the evidence for the presence of presynaptic NMDA receptors at cortical synapses and their possible role in the induction of t-LTD. Two basic models emerge for the induction of t-LTD at cortical synapses. In one model, coincident activation of presynaptic NMDA receptors and CB1 receptors mediates t-LTD. In a second model, CB1 receptors are not necessary, and the activation of presynaptic NMDA receptors alone appears to be sufficient for the induction of t-LTD

Neuromodulation of Spike-Timing-Dependent Plasticity: Past, Present, and Future.

Spike-timing-dependent synaptic plasticity (STDP) is a leading cellular model for behavioral learning and memory with rich computational properties. However, the relationship between the millisecond-precision spike timing required for STDP and the much slower timescales of behavioral learning is not well understood. Neuromodulation offers an attractive mechanism to connect these different timescales, and there is now strong experimental evidence that STDP is under neuromodulatory control by acetylcholine, monoamines, and other signaling molecules. Here, we review neuromodulation of STDP, the underlying mechanisms, functional implications, and possible involvement in brain disorders.BBSR

On the function of somatic and dendritic SK channels in cortical pyramidal neurons

Small-conductance Calcium-activated Potassium (SK) channels play an important role in regulating neuronal excitability. Recent evidence indicates SK channels regulate NMDA receptor activation in dendritic spines and play a role in regulating synaptic plasticity. Using confocal fluorescent Calcium imaging, we investigate the activation of SK channels in spines and dendrites of layer 5 (L5) cortical pyramidal neurons during action potentials (APs). The inhibition of SK channels with apamin results in a location-dependent increase in Calcium influx into dendrites and spines during backpropagating APs (average increase ~40%). This effect was occluded by block of R-type voltage-dependent calcium channels (VDCCs), but not by inhibition of N- or P/Q-type VDCCs, or block of calcium release from intracellular stores. These results indicate that dendritic SK channels regulate Calcium influx into dendrites and spines presumably by regulating the waveform of the backpropagating action potential. In addition, they show that SK channel activation is specifically controlled by Calcium influx through R-type VDCCs, complimenting previous work indicating that SK channels are located within 25-50 nm of their calcium source, in a Calcium nanodomain. SK channels have also long been known to contribute to the medium afterhyperpolarization (mAHP) at the soma for many neuronal types, including L5 pyramidal neurons, where they regulate action potential output gain and the propensity for burst firing. During the experiments using confocal Calcium imaging we noticed that the Calcium indicator used, Oregon Green BAPTA-1, which acts as a fast calcium buffer, blocked the SK mAHP. Subsequent experiments using low concentrations of the slow calcium buffer EGTA (1 mM) produced the same result, suggesting that somatic SK channels are not tightly co-localised with their calcium source. We estimate a coupling distance of greater than 150 nm, suggesting that calcium signaling between somatic SK channels and their calcium source occurs within a microdomain. Consistent with this idea, we show that all known subtypes of VDCCs except R-type were Calcium sources for the apamin-sensitive mAHP at the soma. Spike timing dependent plasticity (STDP) is a form of plasticity whereby the strength of connections between neurons in the brain are modulated following the pairing of postsynaptic APs with presynaptic activity within a narrow time window. The influx of calcium during EPSP-AP pairing is crucial to the induction of STDP. Given we show that dendritic SK channels can be activated by and influence calcium influx during bAPs, we investigated whether dendritic SK channel activation by bAPs can regulate STDP. We show that SK channels activated by bAPs significantly suppress EPSPs at negative timings for L5-L5 synaptic connections by dampening the activation of NMDA receptors. SK channels also suppressed EPSPs in layer 2/3 and hippocampal CA1 pyramidal neurons. Finally, we show that the induction of both LTP and LTD for L5-L5 synaptic connections is constrained by SK channel activation, presumably through their capacity to limit dendritic calcium influx during EPSP-AP pairing. In conclusion, these findings provide new insights into the function of SK channels and the multifaceted role calcium plays in neuronal function

CB1-dependent neuromodulation in the nucleus accumbens core

Tese de Mestrado em Bioquímica, Faculdade de Ciências, Universidade de Lisboa, 2021A Canábis tem sido utilizada pelas populações ao longo dos milénios, tanto para fins medicinais, como para fins recreativos. Apesar da nossa relação de longa data com a planta, os seus efeitos fisiológicos e mecanismos moleculares só foram estudados nas últimas décadas, havendo ainda muito por desvendar. A primeira grande descoberta neste campo foi na década de 1930 com o isolamento de Canabinol (o primeiro derivado da planta), que despoletou o aumento do interesse da comunidade científica pelo potencial medicinal da Canábis. Nos anos noventa, descobriram-se recetores canabinóides do tipo 1 em humanos e pouco depois, o tipo 2 foi também caracterizado. Durante a primeira década dos anos dois mil, a descoberta do primeiro antagonista seletivo do CB1 (Rimonabant) e a produção de ratinhos CB1 knock-out permitiram-nos começar a entender alguns dos efeitos e mecanismos destes recetores. Hoje, sabe-se que ambos os recetores canabinóides podem estar patologicamente expressos em variadíssimos tipos de doenças, como neurológicas (Alzheimer e Esclerose múltipla), psiquiátricas (depressão e esquizofrenia), cardiovascular (Arteriosclerose) e também gastrointestinal (cirrose). Tanto o facto do sistema endocanabinóide estar relacionado com inúmeras patologias, como a descriminalização da Canábis ajudaram a atenuar alguma desconfiança na planta e consequentemente, descobriu-se um novo mundo de possíveis terapias associadas a canabinóides. O sistema endocanabinóide é composto por endocanabinóides, proteínas recetoras e enzimas para a síntese e degradação de endocanabinóides. No cérebro, a sinalização endocanabinóide implica a ativação de CB1, que exerce um importante papel em funções neuronais relacionadas com memória e aprendizagem, controlo motor, sono, entre outros. Em neurónios, CB1 estão acoplados a proteínas Gi/o e encontram-se maioritariamente expressos em membranas pré-sinápticas, particularmente de sinapses Glutamatérgicas. Nestas, a ativação de CB1 e Gi/o medeiam a sinalização retrógrada de endocanabinóides, através da supressão da atividade da enzima Adenil Ciclase que por consequência leva não só à diminuição de cAMP mas como também à inativação de PKA. Desta forma, ocorre o influxo pré-sináptico de Ca2+ e a inibição da libertação de neurotransmissores. Para além da sua expressão em neurónios, os CB1 também estão expressos em astrócitos, onde se encontram acoplados a Gq. A sua ativação leva à cascata MAPK/ERK e regula a excitabilidade neuronal, a transmissão sináptica e plasticidade, ao estimular a libertação de Glutamato. Apesar do mecanismo pré-sináptico da sinalização de endocanabinóides já ter sido extensivamente estudado, alterações pós-sinápticas também podem ocorrer, já que os CB1 conseguem modular a transmissão sináptica mediada através de recetores AMPA e NMDA. É de interesse perceber a relação entre recetores AMPA, NMDA e CB1 pois são essenciais, não só para a transmissão sináptica mas também na plasticidade sináptica. Os recetores AMPA são recetores ionótrópicos de glutamato e estão localizados em terminais pós sinápticos, mais especificamente na densidade pós-sináptica. Quando ativos, contribuem para a abertura de canais iónicos, induzindo assim a despolarização membranar e sendo por isso essenciais para a plasticidade sináptica, aprendizagem e memória. AMPARs são tetraméricos, estando organizados em dois dímeros, cujas subunidades podem variar entre GluA1 – GluA4. Para além de serem permeáveis a Na+, AMPARs sem a subunidade GluA2 são permeáveis a Ca2+, o que possibilita a ativação de cascatas dependentes de Ca2+. Estas cascatas de eventos levam ao trafficking de AMPARs nas membranas pós-sinápticas, processo essencial para a plasticidade sináptica. O aumento de Ca2+ pode também contribuir para a ativação de NMDARs e cascatas MAPK/ERK. Os recetores de NMDA são ionotrópicos e ativados por Glutamato, sendo essenciais para a neurotransmissão excitatória rápida. Tal como AMPARs, as funções de NMDARs nas sinapses são extremamente complexas e diferem entre áreas do cérebro. Esta complexidade é o que permite mecanismos de plasticidade sináptica, não só a iniciação mas também a sua manutenção. NMDARs são tetrâmeros que contêm obrigatoriamente duas subunidades GluN1 e duas subunidades reguladoras, GluN2 ou GluN3. Para além de serem permeáveis a Na+, NMDARs com a subunidade GluN2A têm alta sensibilidade a bloqueios por Mg2+, o que contribui para o influxo de Ca2+. Para além disto, esta subunidade é responsável por controlar os estados aberto/fechado dos canais de NMDAR, o que induz cascatas de CamKII. Estas têm a capacidade de regular o tráfego de NMDARs nas membranas, contribuindo também para a iniciação de cascatas MAPK/ERK. O NAc é um dos principais componentes da via mesocorticolímbica, sendo constituído por duas partes que diferem em morfologia e função – um core e uma shell. Esta zona é responsável não só por emoções como desejo e motivação, mas também emoções associadas ao prazer, como a felicidade e a euforia. Mais especificamente, o core do NAc é responsável pelo processamento cognitivo de funções motoras relacionadas com reforço e recompensa, sendo também responsável pelo vício de Anfetaminas e Cocaína. Para além disto, o NAc está também envolvido em doenças psiquiátricas como a esquizofrenia e a depressão. O NAc é composto por 90% de medium spiny neurons GABAérgicos e o restante corresponde a medium spiny neurons Glutamatérgicos e interneurónios Colinérgicos. Recebe inputs glutamatérgicos do Córtex Pré-frontal, do Hipocampo e da Área Tegmental Ventral, enquanto que os neurónios do output enviam projeções axonais para a Área Tegmental Ventral, a Hipocampo, o Córtex Pré-frontal, entre outros. Os circuitos e neurotransmissores do NAc já foram extensivamente estudados, contudo ainda existem muitas questões quanto à influência do CB1, recetor que já mostrou ser abundante nesta região, particularmente em terminais glutamatérgicos de corpos celulares GABAérgicos. Para além disto, já foi mostrado que a ativação farmacológica de CB1 diminui a transmissão glutamatérgica evocada, sugerindo que estes têm um papel fisiológico fundamental na excitabilidade do NAc. Os mecanismos pré-sinápticos já foram estudados, contudo, alterações pós-sinápticas também são possíveis já que os CB1 podem modular AMPARs e NMDARs. É importante perceber a relação entre CB1, AMPARs e NMDARs já que estes recetores são essenciais para a transmissão e plasticidade sináptica, afetando os outcomes comportamentais. De forma a investigar se a ausência crónica de CB1 afeta a transmissão glutamatérgica no núcleo do NAc, foi utilizada eletrofisiologia whole-cell patch clamp ex vivo combinada com farmacologia e linhas de ratinho transgénicas. Primeiro, verificou-se que a deleção total de CB1 afeta a transmissão sináptica espontânea ao aumentar a frequência de eventos sinápticos. Estes resultados sugerem que CB1 são essencias para controlar a libertação e clearence de Glutamato. De seguida, verificou-se que os endocanabinóides não são tónicamente libertados no core do NAc, sugerindo que os resultados anteriormente são causados pela falta crónica de CB1. Em terceiro, verificamos que não existem alterações no ratio de AMPA/NMDA, sugerindo que podem existir alterações nas subunidades de recetores AMPA e NMDA. Depois, verificou-se que a deleção total de CB1 prejudica a atividade de AMPARs permeáveis a Ca2+ em medium spiny neurons no core do NAc, sugerindo que os mecanismos de tráfego podem estar afetados. Por último verificou-se que a deleção total de CB1 aumenta a presença da subunidade GluN2A em NMDARs no core do NAc, sugerindo que pode estar a compensar a falta de CB1. Para além disto, pode também haver um aumento crónico de PKA, PKC ou CaMkII. Com este projeto podemos então concluir que a ausência de CB1 induz alterações na atividade sináptica que permitem o aumento da libertação de glutamato. A ausência de CB1 também causa alterações pós-sinápticas ao modificar o tipo de subunidades presente nos recetores AMPA e NMDA. Seria interessante perceber se estas alterações são diretamente causadas pela ausência de CB1 ou por outro mecanismo. Para além disto, devemos também tentar perceber como é que estas alterações afetam a maturação sináptica e os mecanismos de plasticidade.The Endocannabinoid System (ECS) is mainly composed by endocannabinoids (eCBs) and cannabinoid receptor proteins. The endocannabinoid signaling plays a major role in neural functions, regulating emotional and motivational states mostly through the activation of Cannabinoid type-1 receptors (CB1), the main effectors of the ECS in the brain. The Nucleus Accumbens (NAc) is a major component of the Mesocorticolimbic pathway, being a key structure in mediating emotional and motivation processing, modulating reward and also pleasure. The NAc core receives glutamatergic inputs and as CB1 have been shown to be essential to maintain evoked glutamatergic transmission, it suggests that they play a relevant physiological role for the NAc core excitability. Moreover, manipulation of CB1 signaling within this brain region triggers robust emotional/ motivational alterations related to drug addiction and other psychiatric disorders (CB1 expressing neurons in the Nac, 2012). Although the associated presynaptic mechanism of endocannabinoid signaling has already been studied, postsynaptic changes may occur, as CB1 are able to modulate AMPAR and NMDARmediated synaptic transmission. It is of interest to understand the relationship between AMPA, NMDA and CB1 receptors in the NAc because they are essential not only to synaptic transmission but also plasticity, which can affect certain behaviors. Using ex vivo whole-cell patch clamp electrophysiology combined with pharmacology and transgenic mouse lines, we aimed at investigating whether the chronic lack of CB1 affects spontaneous and evoked glutamatergic transmission in the NAc core. Our results show that full CB1 knock-out mice (CB1 -/- ) have an increased frequency of miniature synaptic events without changes in their amplitude, while blocking CB1 with the antagonist Rimonabant shows no effect. Moreover, CB1 -/- lack Calcium-permeable AMPARs and have an increase in GluN2A-containing NMDARs. Our results confirm the CB1 presynaptic mechanism of action but also suggest a complementary postsynaptic mechanism. Altogether these results show that the chronic lack of CB1 is able to induce postsynaptic changes in medium spiny neurons (MSNs) from the NAc core, specifically in AMPAR and NMDAR subunit composition