Layer- and column-specific knockout of NMDA receptors in pyramidal neurons of the mouse barrel cortex

Viral vectors injected into the mouse brain offer the possibility for localized genetic modifications in a highly controlled manner. Lentivector injection into mouse neocortex transduces cells within a diameter of approximately 200mum, which closely matches the lateral scale of a column in barrel cortex. The depth and volume of the injection determines which cortical layer is transduced. Furthermore, transduced gene expression from the lentivector can be limited to predominantly pyramidal neurons by using a 1.3kb fragment of the alphaCaMKII promoter. This technique therefore allows genetic manipulation of a specific cell type in defined columns and layers of the neocortex. By expressing Cre recombinase from such a lentivector in gene-targeted mice carrying a floxed gene, highly specific genetic lesions can be induced. Here, we demonstrate the utility of this approach by specifically knocking out NMDA receptors (NMDARs) in pyramidal neurons in the somatosensory barrel cortex of gene-targeted mice carrying floxed NMDAR 1 genes. Neurons transduced with lentivector encoding GFP and Cre recombinase exhibit not only reductions in NMDAR 1 mRNA levels, but reduced NMDAR-dependent currents and pairing-induced synaptic potentiation. This technique for knockout of NMDARs in a cell type, column- and layer-specific manner in the mouse somatosensory cortex may help further our understanding of the functional roles of NMDARs in vivo during sensory perception and learning.

Analysis of Neuronal Microcircuits in L2/3 of Mouse Barrel Cortex

Michael Avermann

Neocortical microcircuits are thought to contribute importantly to the sophisticated processing abilities of the mammalian brain. Neurons within the neocortex communicate with each other through releasing the excitatory neurotransmitter glutamate or the inhibitory neurotransmitter GABA. In order to begin to understand the functional operation of the neocortex, it will clearly be important to establish the basic organizing principles of synaptic connectivity between different classes of cortical neurons. Here I specifically investigate excitatory and GABAergic neuronal networks within layer 2/3 of mouse somatosensory cortex in vitro. In order to analyze synaptic connectivity I made multiple simultaneous whole-cell recordings from GFP expressing GABAergic neurons and from GFP-negative pyramidal neurons targeted by two-photon microscopy. Additionally, I express channelrhodopsin-2 (ChR2), a light-activated cation channel, in genetically defined populations of excitatory neurons which allows optically control of neuronal networks with millisecond precision. I show that optically evoked, near synchronous action potentials in excitatory neurons are sufficient to drive rapid and reliable disynaptic local inhibition onto neighboring pyramidal neurons. Local inhibition was mediated by parvalbumin expressing, fast-spiking (FS) GABAergic neurons, which responded to excitatory network input with significantly larger amplitude depolarizing PSPs compared to both non-fast-spiking (NFS) GABAergic and excitatory pyramidal neurons. The strong excitatory drive onto FS neurons can partially be explained with the finding that pyramidal neurons in cortical layer 2/3 synaptically connect to FS neurons with a higher probabilities and larger unitary EPSPs. Furthermore, FS neurons provided strong unitary inhibitory feedback onto pyramidal neurons and other FS neurons in layer 2/3. These results suggest an important role of FS GABAergic neurons in mediating strong local inhibition to control the extent of excitatory layer 2/3 network activity.

EPFL2011

Layer-Dependent Short-Term Synaptic Plasticity Between Excitatory Neurons in the C2 Barrel Column of Mouse Primary Somatosensory Cortex

Sandrine Lefort, Carl Petersen

Neurons process information through spatiotemporal integration of synaptic input. Synaptic transmission between any given pair of neurons is typically a dynamic process with presynaptic action potentials (APs) evoking depressing or facilitating postsynaptic potentials when presynaptic APs occur within hundreds of milliseconds of each other. In order to understand neocortical function, it is therefore important to investigate such short-term synaptic plasticity at synapses between different types of neocortical neurons. Here, we examine short-term synaptic dynamics between excitatory neurons in different layers of the mouse C2 barrel column through in vitro whole-cell recordings. We find layer-dependent short-term plasticity, with depression being dominant at many synaptic connections. Interestingly, however, presynaptic layer 2 neurons predominantly give rise to facilitating excitatory synaptic output at short interspike intervals of 10 and 30 ms. Previous studies have found prominent burst firing of excitatory neurons in supragranular layers of awake mice. The facilitation we observed in the synaptic output of layer 2 may, therefore, be functionally relevant, possibly serving to enhance the postsynaptic impact of burst firing.

Oxford Univ Press Inc2017

Structure and dynamics of the neocortical microcircuit connectivity

Jean-Vincent Le Bé

The neocortex is the most computationally advanced portion of the brain. It is currently assumed to be composed of a large number of "cortical columns" – intricate arrangements of cortical neurons approximately 300-500 µm in diameter and 2-5 mm in height in humans – that might serve as the elementary computational unit of the neocortex. Understanding the computation performed by this microcircuit is one of the keys to our comprehension of the brain. The so-called cortical column is not a static entity, however, and it evolves throughout a lifetime and continually adapts to the information from its cortical environment. Despite the differences between cortical columns across the cortex, a number of common features have been identified: a laminar structure, the dynamics of connections between identified neurons or the mechanisms for these connections to be modified (that give its specificity to each microcircuit). This thesis presents the description of the differential connectivity and synaptic dynamics across cell populations and the long term neuronal rewiring in a particular neuronal population within the cortical column. Somatic whole cell recordings have been performed to probe the connectivity, synaptic dynamics and plasticity of the connections in the rat neocortex. Two populations of layer V pyramidal neurons were studied in particular: cortico-callosally projecting pyramidal cells (CCPs) and thick tufted pyramidal cells (TTCs). The first major results from this work revealed the degree of connectivity and the linear dynamics of the CCPs population when compared to the TTCs. CCPs have nearly 4 times fewer interconnections and subsequent post-synaptic potentials were less decreasing in amplitude along a pre-synaptic series of action potentials. Long term configuration of TTC networks was explored. These experiments show for the first time the emergence of new functional synaptic connections between TTCs within hours. Activation of the slice by glutamate greatly increases their rate of emergence and this work demonstrated that metabotropic glutamate receptor 5 (mGluR5) activation and action potential firing are required for new connections to be formed in this experimental protocol. Newly formed connections respond in a more linear fashion and have weaker post-synaptic influence than already existing connections. Pre-existing connections are also modified after stimulation, requiring mGluR5, action potentials as well as α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) and N-methyl-D-aspartic acid (NMDA) receptors activation. The activation of group III metabotropic glutamate receptors (mGluRs) however results in a decrease in the strength of connections. Finally, the influence of inhibitory interneurons on the activity and connectivity between TTCs was also investigated. The results of this study show that firing of inhibitory interneurons can be triggered by the input of only one pyramidal cell. They further show that stimulation of a single TTC can result in an hyperpolarization of the post-synaptic TTC mediated by an interneurone. When the pre-synaptic neuron is also directly connected to the post-synaptic neuron with an excitatory synapse, the indirect inhibitory connection serves to curtail the excitatory response. This work has provided a new insight into the dynamic nature of the cortical microcircuitry, showing that it evolves rapidly and can adapt, reconfigure and rewire itself in remarkably short time-spans. It also describes the variety of dynamics exhibited by the different types of pyramidal cells, due to either the projecting site specificity or to the action of an intermediate interneuron.