El núcleo posterior medial del tálamo y su implicación en los procesos perceptivos y cognitivos

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Anatomía, Histología y Neurociencia. Fecha de lectura: 11-05-2021Se desconoce en gran medida cómo el sistema somestésico extrae información del flujo de señales sensoriales. La información procedente de las vibrisas es procesada principalmente por dos vías ascendentes paralelas hacia la corteza cerebral. Sin embargo, se desconoce la implicación funcional de las diferentes estructuras que componen dichas rutas. Mediante la combinación de técnicas electrofisiológicas y farmacológicas in vivo en ratas, encontramos diferencias significativas entre estas vías. Aunque está bien asumido que el POm y el VPM responden a la estimulación de las vibrisas contralaterales, encontramos que el primero es capaz de responder también a las ipsilaterales. Mediante la integración de señales simultaneas procedentes de vibrisas en ambos lados de la cara, está implicado en la representación de eventos táctiles bilaterales. Esto demuestra la implicación de los núcleos talámicos sensoriales de tipo 'higher-order' en la percepción bilateral. Encontramos que los núcleos POm están mutuamente conectados a través de la corteza formando un bucle o 'loop' funcional. Revelamos la naturaleza y el contenido de los mensajes transmitidos a través de este circuito mostrando que dichos mensajes son 'patrones estructurados de actividad sostenida'. Estos mensajes son transmitidos preservando su estructura integrada. La implicación de diferentes áreas fue investigada descubriendo que S1 juega un papel protagonista en dicho 'loop' POm-POm. También encontramos diferente implicación laminar en esta área en el procesamiento de actividad sostenida y en su transmisión entre hemisferios. Proponemos un modelo teórico en el que dichos 'patrones estructurados de actividad sostenida' generados por el POm pueden jugar un papel relevante en las funciones perceptivas, motoras y cognitivas. Además, demostramos que el POm está involucrado en la representación de patrones sensoriales complejos. Este núcleo es muy sensible a la activación simultanea de las vibrisas y a las complejas interacciones espaciotemporales que se producen entre ellas. La estructura espaciotemporal de dichos patrones y la complejidad de sus partes son reflejados en precisos cambios de actividad en el POm. Nuestros resultados sugieren que este núcleo podría ser un codificador general de patrones. La naturaleza (estructurada versus discreta), el tipo (sostenido versus transitorio) y el contenido (integrado versus segregado) de la actividad neural procesada y transmitida por estos núcleos determina su implicación funcional y puede permitir clasificarlos. Proponemos la hipótesis de los Componentes Complementarios para explicar estas diferencias. Además, revelamos la capacidad del POm para ajustar el procesamiento en las cortezas S1 y S2 mediante la inducción de una precisa inhibición en determinadas capas corticales. Esta modulación está mediada por neuronas GABAérgicas de la capa 1. La hipótesis de Computación Cortical por Resultados Discretos propuesta aquí puede explicar la implicación funcional de dicho ajust

All-optical interrogation of neural circuits during behaviour

This thesis explores the fundamental question of how patterns of neural activity encode information and guide behaviour. To address this, one needs three things: a way to record neural activity so that one can correlate neuronal responses with environmental variables; a flexible and specific way to influence neural activity so that one can modulate the variables that may underlie how information is encoded; a robust behavioural paradigm that allows one to assess how modulation of both environmental and neural variables modify behaviour. Techniques combining all three would be transformative for investigating which features of neural activity, and which neurons, most influence behavioural output. Previous electrical and optogenetic microstimulation studies have told us much about the impact of spatially or genetically defined groups of neurons, however they lack the flexibility to probe the contribution of specific, functionally defined subsets. In this thesis I leverage a combination of existing technologies to approach this goal. I combine two-photon calcium imaging with two-photon optogenetics and digital holography to generate an “all-optical” method for simultaneous reading and writing of neural activity in vivo with high spatio-temporal resolution. Calcium imaging allows for cellular resolution recordings from neural populations. Two-photon optogenetics allows for targeted activation of individual cells. Digital holography, using spatial light modulators (SLMs), allows for simultaneous photostimulation of tens to hundreds of neurons in arbitrary spatial locations. Taken together, I demonstrate that this method allows one to map the functional signature of neurons in superficial mouse barrel cortex and to target photostimulation to functionally-defined subsets of cells. I develop a suite of software that allows for quick, intuitive execution of such experiments and I combine this with a behavioural paradigm testing the effect of targeted perturbations on behaviour. In doing so, I demonstrate that animals are able to reliably detect the targeted activation of tens of neurons, with some sensitive to as few as five cortical cells. I demonstrate that such learning can be specific to targeted cells, and that the lower bound of perception shifts with training. The temporal structure of such perturbations had little impact on behaviour, however different groups of neurons drive behaviour to different extents. In order to probe which characteristics underly such variation, I tested whether the sensory response strength or correlation structure of targeted ensembles influenced their behavioural salience. Whilst these final experiments were inconclusive, they demonstrate their feasibility and provide us with some key actionable improvements that could further strengthen the all-optical approach. This thesis therefore represents a significant step forward towards the goal of combining high resolution readout and perturbation of neural activity with behaviour in order to investigate which features of the neural code are behaviourally relevant

Mechanisms underlying postnatal development of primary somatosensory cortex

Layer IV of the mouse somatosensory cortex contains discrete cytoarchitectonic units called 'barrels', formed in response to thalamocortical axon invasion by two processes: translocation of cortical cells to form a cell-dense barrel wall and cell-sparse barrel hollow, and selective dendritic elaboration toward thalamocortical afferents to form oriented dendritic branch patterns. Interestingly gene knockout of several members of the N-Methyl-D-Aspartate (NMDA) receptor -complex (NRC) disrupts barrel formation, indicating that synaptic activity is critical for barrel formation. Little is known of the cellular processes initiated by glutamate receptor activation; however, recent evidence suggests an interaction between neuronal activity and Wnt signalling. Wnts are secreted glycoproteins, are powerful regulators of cell proliferation and differentiation, and their signalling pathway involves proteins that directly participate in both gene transcription and cell adhesion. Wnt7a and Dv11 knockout mice exhibit delays in glomerular rosette formation; a cerebellar structure similar to barrels whose development involves granule cell migration and dendritic rearrangement. Furthermore activity dependent Wnt release can regulate the enhancement of dendrite arborisation, raising the possibility that NRC components and Wnts may interact to regulate barrel development.Recent findings suggest that membrane association guanylyl kinases (MAGUKs) may be the key scaffolding molecules that mediate the interaction between glutamate receptor and Wnt signalling pathways. The MAGUK family includes Postsynaptic Density (PSD)-95 and Synapse Associated Protein (SAP)-102, two key molecules of the NRC during barrel formation. These MAGUKs also bind the Wnt receptor family Frizzled and SAP-102 binds to APC, a key Frizzled-signalling protein. As a first step in examining a role for Wnts in barrel formation, the gene expression patterns of members of the Wnt, Frizzleds and secreted Frizzled related protein (sFRPs) families during barrel cortex development were measured using degenerate primer RT-PCR, quantitative real-time PCR and in-situ hybridization. Wnts 2b, 3, 4, 5a, la, 7b, 9a, 11, 16 were found in the barrel cortex, suggesting that these members of the Wnt family may play a role in barrel development, and Wnt7b, Frizzled 4, Frizzled 9, and Frizzled 3 were conspicuously downregulated in mutant mice that lack barrels, namely Plc-ß1⁻/⁻, Pkar2ß⁻/⁻ and Mglur5⁻/⁻ mice.In order to determine whether Wnts, members of the Wnt signaling machinery and MAGUKs associated with Wnt signaling are essential for barrel formation, the barrel phenotype of all available postnatally viable Wnt gene knockouts, Wnt2b⁻/⁻, Wnt7a⁻/⁻, Wnt8b⁻/⁻, Wnt signaling component knockouts Dvl⁻/⁻, MAGUK knockouts Sap-102'1', Psd-95'1' and double knockouts of Wnt7a"Dvr'~ and of Sap-102⁻/⁻Psd-95⁻/⁻ were examined. Barrels appeared normal in all mutants, apart from compound Sap-102⁻/⁻Psd-95⁻/⁻ and Sap-102⁻/⁻Psd-95⁻/⁻ mice, which exhibited poorer barrel segregation compared to wild type.In order to achieve a detailed understanding of the mechanisms by which neuronal activity regulates barrel development, we need a detailed understanding of the intracellular pathways activated by NMDA receptors during development. In searching the literature, data concerning the developmental expression patterns of NRC components can be difficult to locate, as the prevailing database tools used either search only title, author and keyword abstract text (NCBI PubMed) potentially missing desired information, or as in the unique case of Google Scholar, search the full text of electronically published papers, but yield overwhelming numbers of results in the process.The Jackson Laboratories MGI suite offers an impressive way of mining the literature for such data, but the content is sparse, relying on author submission and attempting to map expression throughout the whole mouse. For example, the only gene present in MGI for postnatal layer IV cerebral cortex is Apc2.In order to bring together the data in the literature and from the mouse genome projects into a usable and accessible way, we decided to create a web-based centralised resource for the developmental neuroscience community, containing expression profiles of NRC components within mouse somatosensory cortex. By performing exhaustive literature searches utilising Google Scholar and PubMed, and linking to sequence and mutant mouse availability information elsewhere, BGI offers a portal for such information and also offers a forum for the notification of unpublished observations of transgenic animals displaying normal barrel formation, preventing duplication of experiments. Barrelgene.info should provide a key resource for any researcher interested in the molecular basis of cortical development

Calcium dynamics in dendrites and spines of spiny neurons in the somatosensory ‘barrel’ cortex of the rat

Two-photon excitation fluorescence microscopy was combined with the patch-clamp technique to study the Ca2+ dynamics in dendrites and spines of spiny neurons of layer 4 of the somatosensory cortex in acute thalamocortical brain slices of young (P13-P15) rats. Back-propagating action potentials (bAPs) resulted in a transient rise in Ca2+ in all dendrites and spines tested, representing a global intracellular chemical signal about the activity of the cell. In contrast, synaptically evoked excitatory postsynaptic potentials (EPSPs) resulted in a synapse specific, local increase in Ca2+. Pairing both stimuli at different inter-stimulus intervals revealed a precisely tuned coincidence detection mechanism for pre- and postsynaptic activity, coded in the peak Ca2+ transient amplitude. Linear, sub- and supralinear summation of the Ca2+ transients, depending on the time interval and the order of bAP and EPSP, was found. Ca2+ influx was maximal when the action potential followed synaptic stimulation within less than 20 ms. The mechanism of maximal Ca2+ influx could be explained by the properites of the NMDA receptor channel, which was activated by binding glutamate during synaptic stimulation and subsequent relief of the Mg2+ block by the bAP. Coincidence detection was restricted to the synaptic contact and it did not depend on the distance of the contact from the soma. This temporally and spatially highly restricted coincidence detection mechanism, which emplyed the Ca2+ transient amplitude as a readout signal might serve as an input specific trigger for spike-timing dependent plasticity. Indeed potentiation of EPSPs to 150% of the baseline amplitude could be induced by pairing extracellular stimulation with bAPs within the coincidence detection interval. Reversing the order of the stimuli resulted in depression of the EPSP amplitude to 70%. Thus it was concluded that spiny neurons in layer 4 of the juvenile rat barrel cortex exhibit spike-timing dependent plasticity, which corresponded well to the Ca2+ code used by their spines for coincidence detection

GABA signaling in the thalamus

Inhibition of neuronal activity in networks of the mammalian central nervous system is essential for all fundamental brain functions, ranging from perception, to consciousness, to action. Both exacerbation and diminution of inhibition dramatically affect our behavioral capacities, indicating that, in the healthy brain, strength and dynamics of inhibition must be precisely balanced. Inhibitory functions are primarily accomplished by neurons releasing the neurotransmitter GABA. According to their wide variety of functions, GABAergic neurons show a tremendous diversity in morphological, biochemical and functional characteristics. The combination of these diverse properties allows the brain to generate interneurons acting as, for examples, filters, co-incidence detectors or contrast enhancers. GABAergic signaling in thalamus plays an essential role in controlling sensory information flow from the periphery to the cortical processing centers, and in generating sleep-related neuronal rhythms. Surprisingly, however, the diversity of GABAergic neurons is remarkably limited in thalamic networks. Both functions mentioned have been tightly associated with two homogeneous groups of GABAergic neurons arising within thalamic nuclei or within the nucleus reticularis, a shell of inhibitory nuclei surrounding the dorsal thalamus. The results arising from the present thesis challenge the view that the diversity of GABAergic signaling in thalamus is comparatively limited and proposes that, to fully understand GABAergic signaling in thalamus, at least two additional aspects have to be considered. First, it shows that GABAergic signaling arising from the nucleus reticularis can have a profound effect on the synthesis of second messenger compounds that are important in the control of neuronal rhythmicities and in the statedependent control of gene expression. Second, it demonstrates the functional relevance of a previously undescribed extrathalamic and extrareticular inhibitory pathway that arises within the anterior pretectal nuclei, indicating that the architecture of GABAergic signaling in thalamus has to be complemented by a conceptually novel, powerful afferent pathway. The first part investigates the modulation of cAMP synthesis by GABA in thalamocortical neurons through the activation of the Gi-coupled GABAB receptors. GABAB receptors can provide two different cAMP signals in the neurons. First, GABAB receptor activation depresses the level of cAMP inside thalamocortical neurons. However, a large and long cAMP signal is observed when GABAB receptors are activated concomitantly with b-adrenergic receptors, which are Gscoupled receptors. In the presence of GABAB receptor agonists, the moderate cAMP increase produced by b-adrenergic receptor activation is transformed into a large synthesis of cAMP. Remarkably, the activation of the GABAB receptors at the synapses between reticular neurons and thalamocortical neurons also potentiates the effects of b-adrenergic receptors. Thus, GABAB receptors modulate cAMP signals at synapses that are important for the regulation of the state of arousal. The second part provides the first electrophysiological description of synaptic connections between the anterior pretectum group and the thalamic higher-order nuclei. Electric stimulation in the anterior pretectum group evoked inhibitory postsynaptic responses (IPS) in the thalamocortical neurons of the higher-order nuclei. We showed that the IPS responses were mediated via the GABAA receptors activated through monosynaptic connections between the APT and the higher-order nuclei. Functionally, the anterior pretectum modulated the discharge properties of the thalamocortical neurons, suggesting an important role of this nucleus in the dialogue between the thalamus and the cortex

Bioinformatics and genetics analysis of experience dependent plasticity in the mouse barrel cortex

Formation of neuronal circuits represent memories, making synaptic plasticity the root of learning and memory (Buonomano and Merzenich 1998). Neuronal plasticity has been studied using facial vibrissae deprivation paradigm in rodents (Fox 1992). Whisker deprivation alters the balance of activity in cortical neurons and their responses to sensory input, providing good grounds to study experience dependent plasticity (Simons and Land 1987 Fox 1992). Alterations in gene expression underpinning changes in cortical activity have been investigated in this thesis. The molecular signature underlying the temporal effect of repeated anaesthesia was identified and provided a fertile area for future work, revealing the necessity to separate anaesthesia from deprivation induced changes. Changes in gene expression were gender specific, with the females exhibiting quicker neuronal organisation. Taking under consideration the two confounding factors anaesthesia and gender, a new normalisation protocol was developed underpinning investigations of plasticity dependent transcriptional alterations. The present study confirmed the two molecular mechanisms underlining synaptic plasticity (Shi et al. 1999) with early time points (Day 1) revealing alterations of existing synaptic proteins and later time points (Day 8 and 16) indicating neurotransmitter release regulating gene expression. Day 8 was identified as the critical time point for plasticity, exhibiting the peak of transcriptional changes. Gender specificity was evident, indicating a role for hormonal-dependent gene expression, which future studies should consider. Ontological analysis has confirmed the role of Ca2+ trafficking (via AMPARs and NMDARs) and calcium dependent binding (involving molecules like Calmodulin) in a variety of pathways, such as transporter activity, channel activity and neurogenesis, associated with gene transcription and regulation of plasticity. A significant up-regulation of the expression profiles of transcripts associated with plasticity, NOS1, NOS3 and Bassoon was observed at Day 8 in wild type mice. GluRl-/- mice revealed the direct relationship of these genes with the GluR 1 subunit of AMPA receptors. A delayed up-regulation was detected after 16 days, suggesting a plausible delayed compensatory mechanism in the absence of the GluRl subunit of the AMPA receptor. Gene ontology provided a functional footprint for plasticity even in the GluRl-/- mice, known to exhibit impaired post-synaptic plasticity (Schmitt et al. 2005)